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403

16RNA Interaction with Small Ligandsand Peptides

Joseph D. PuglisiDepartment of Structural BiologyStanford University School of MedicineStanford, California 94305-5400

James R. WilliamsonDepartment of Molecular Biology andThe Skaggs Institute of Chemical BiologyThe Scripps Research InstituteLa Jolla, California 92037

RNA is able to bind small molecule (MW <2000) ligands. These ligandscan be drugs that bind to sites in biological RNAs, peptide fragments oflarger proteins, or molecules for which RNA-binding sites (aptamers)have been selected by in vitro evolution. The RNAs that bind smallmolecules are modular, and the high local thermodynamic stability ofRNA often assures stable folding of RNA domain fragments. Therefore,RNA–ligand interactions can often be studied using drastically reducedsystems. RNA oligonucleotides can be produced in large quantities, andadvances in NMR spectroscopy have allowed structure determination ofRNA by NMR (Varani and Tinoco 1991; Chang and Varani 1997; Puglisiand Puglisi 1998). RNA–small molecule ligand complexes are particu-larly amenable to NMR structure determination.

This review focuses on the large number of NMR structures ofRNA–ligand complexes that have been determined in recent years. Thesestructures have revealed general themes for ligand recognition and haveprovided insights into the biological functions of RNAs. The binding andmanipulation of small-molecule substrates was probably a central featureof the RNA World.

STRUCTURAL STUDIES OF RNA APTAMERS

Three RNA aptamers whose structures have been determined at highresolution using multidimensional heteronuclear NMR (Feigon et al.

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404 J.D. Puglisi and J.R. Williamson

1996): ATP (Dieckmann et al. 1996; Jiang et al. 1996), FMN (Fan et al.1996), and theophylline (Zimmermann et al. 1997), are shown in Figure1. For comparison, the complex of HIV TAR with argininamide (Puglisiet al. 1992; Aboul-ela et al. 1995) is included as a small ligand–RNAcomplex, although this structure is not an aptamer. The structure of two

Figure 1 Secondary structures of RNA aptamers discussed in the text. (a) FMNaptamer; (b) ATP aptamer; (c) theophylline aptamer; (d) HIV TAR RNA. Nu-cleotides that are conserved in the selected aptamer are colored: blue are con-served nucleotides without non-Watson-Crick hydrogen binding, green are con-served nucleotides with non-Watson-Crick hydrogen bonding, and magenta areconserved nucleotides with direct hydrogen bonds to the ligand.



RNA Ligand Interactions 405

other aptamers that bind arginine and citrulline have been studied byNMR (Yang et al. 1996), but the role of many of the conserved sequenceelements in these two structures is not yet clear, and these are not dis-cussed here.

Aptamer sequences are selected from pools of random-sequenceRNAs by repeated passage over a column derivatized with the desired li-gand (Gold et al. 1995). After each passage, the RNAs bound to the col-umn are eluted and amplified by a reverse transcription-PCR-transcriptionprocedure (see Chapter 5). After the last round, the RNA pools are cloned,and individual clones are sequenced. Analysis of the sequences in allcases yielded core sequences for the aptamer that were highly conserved(Fig. 1). Each core sequence was flanked by helical regions composed ofWatson-Crick base pairs whose sequence is not critical for small-molecule recognition. For all three of these aptamer sequences, it was pos-sible to construct a minimal aptamer that was sufficiently small to permitNMR structure determination and retained high-affinity ligand interaction(Kd < 10–5 M) .

On the basis of our present knowledge and understanding of RNAstructure, it is not possible to predict from these secondary structures whatthe three-dimensional structures might be. Fortunately, it is now possibleto determine these structures by NMR spectroscopy, as described below.

The FMN Aptamer

FMN (Flavin mononucleotide) is a nucleotide cofactor found in en-zymes. The secondary structure of the FMN aptamer (Burgstaller andFamulok 1994) (Fig. 1a) is composed of an asymmetric internal loop ofsix nucleotides opposite five nucleotides. All of the nucleotides in theinternal loop except one are purines. The schematic structure of thebases in the conserved region is shown in Figure 2a. A continuous stackof purines is formed on one strand from A23 to G28, and all of thesepurines are involved in hydrogen-bonding interactions, primarily withthe opposite strand of the internal loop. G24, A25, G27, and G28 allform non-Watson-Crick base pairs across the helix, and A26 forms hy-drogen bonds to the flavin heterocycle. A base triple structure is formedbetween A25, U12, and G10, and this forms a platform on which theflavin heterocycle is stacked; the stacking continues through G9 and A8.The FMN ligand is effectively sandwiched between stacked purines, asshown in Figure 3a, and specific hydrogen bonds supplied by A26 pro-vide additional stability.




The ATP Aptamer

RNA aptamers that are highly specific for binding ATP (adenosinetriphosphate) over GTP have been selected (Sassanfar and Szostak 1993).The ATP aptamer, which also binds AMP with high affinity, contains anasymmetric internal loop of 11 nucleotides opposite a single nucleotide(Fig. 1b). In contrast to the FMN aptamer, several of the nucleotides inthis loop are not conserved in the selected pools. In the structure of theRNA–AMP complex (Dieckmann et al. 1996; Jiang et al. 1996), it is re-vealed that these nucleotides either provide a stacking scaffold or theyact as spacers to connect the essential structural elements (Fig. 2b). Theadenine base of AMP is stacked between two conserved purines and ishydrogen-bonded by two other conserved purines in the asymmetricinternal loop, as shown in Figure 3b. The two A-form helical segments inthe ATP aptamer are oriented nearly perpendicular to each other, and eachhelix is capped by a G-G base pair. The structure formed by G8, A9, A10,and the bound adenine base is very similar to the structure found inGNRA tetraloops (Heus and Pardi 1991). This U-turn motif is combinedwith other hydrogen-bonding and stacking elements to form the essentialrecognition element.

Figure 2 Schematics of the tertiary structures for RNA–aptamer complexes. (a)FMN aptamer; (b) ATP aptamer; (c) theophylline aptamer; (d) HIV TAR RNA.Nucleotides are colored as in Fig. 1; the ligand is shown in orange. Stacking isshown by black bars, hydrogen bonding by dashed lines.




Figure 3 Three-dimensional structures of the RNA–aptamer complexes dis-cussed in the text. (a) FMN aptamer; (b) ATP aptamer; (c) theophylline aptamer;(d ) HIV TAR–arginine complex. The same coloring scheme as in Figs. 1 and 2is used.




The Theophylline Aptamer

RNA aptamers have been selected that discriminate between theophyllineand caffeine, two planar aromatic purine derivatives that differ by a sin-gle methyl group (Jenison et al. 1994). The secondary structure of thetheophylline aptamer is shown in Figure 1c, and the schematic of the basearrangements in the structure (Zimmermann et al. 1997) is shown in Fig-ure 2c. The theophylline is bound by a complex network of base-stackingand hydrogen-bonding interactions. The central element of the bindingsite is a stack of three base-triple interactions. The central triple of thisstack is formed by hydrogen bonding between the theophylline base andtwo conserved nucleotides, and the outer triples are formed by three ofthe conserved nucleotides. These interactions completely surround the li-gand by stacking interactions from above and below, and with hydrogen-bonding interactions at the sides (Fig. 3c). The central triple core is sup-ported by other non-Watson-Crick interactions. However, the stackinginteractions follow an unusual nonadjacent pattern, with bases U6 and A7forming an intercalated stack with C21 and C22. The entire conservedcore structure is an intricate laminar network of stacking and hydrogen-bonding interactions.

The TAR–Arginine Complex

The TAR element is the binding site for the HIV Tat protein. The portionof the Tat protein involved in binding contains a number of arginines, andit has been shown that the guanidinium group of an arginine derivative,argininamide, will specifically bind to the TAR element (Tao and Frankel1992). The secondary structure of TAR is shown in Figure 1d, and the basesimportant for Tat and argininamide binding are indicated. In contrast to thehigh affinities of the aptamer-ligand complexes, argininamide has a weakaffinity for TAR RNA (Kd = ~10–3 M). In the HIV TAR–argininamidecomplex (Puglisi et al. 1992, 1993; Aboul-ela et al. 1995; Brodsky andWilliamson 1997), the side-chain guanidinium group makes a pair of hy-drogen bonds with the N7 and O6 of G26 (Fig. 2d), as well as electrostaticcontacts with phosphate groups. Argininamide binding induces a confor-mational change in TAR RNA (Aboul-ela et al. 1995). In the free RNA, thepyrimidine bulge is stacked between the two helical stems, with distortioninduced in the width of the helical major groove. On binding, the bulge nu-cleotides are extruded from between the stems, and U23 is positioned in themajor groove near A27 (Fig. 3d). A large body of evidence (Puglisi et al.1992, 1993; Brodsky and Williamson 1997) suggests that U23 forms a




base-triple interaction with A27-U38, with the arginine side chain posi-tioned below the U23 base. The base-triple interaction stabilizes the RNA-binding pocket for the arginine side chain. Interaction of the delocalizedelectrons in the guanidinium group with the uracil base may also stabilizethe RNA–amino acid interaction through van der Waals interactions.

Evolutionary Pressure on Aptamers

The three aptamer–ligand complex structures described above provide atantalizing view of the versatility of RNA structure. However, these RNAsequences are completely nonbiological, and there is no clear role for suchRNA–small molecule complexes in cellular processes. The aptamer se-quences were usually identified as rare members from very large randompools of sequences. In addition, a biological selection pressure for thesemotifs does not clearly exist. Although such RNA motifs may have beenfunctional in ancient RNA machines, their presence in current organismsis unsubstantiated.

Interestingly, all of the aptamer sequences are not well-structured inthe absence of their ligands, and formation of the complicated RNA struc-ture is induced by ligand binding. In contrast, TAR RNA adopts a similarsecondary structure in the presence and absence of argininamide, andstructural rearrangements only involve bulge-stem base-triple formation.The aptamer RNA sequences are under no pressure during the in vitro se-lection process to form a particular structure in the absence of ligand. Theonly requirements for propagation during the selection are binding to theaffinity matrix, elution from the affinity matrix, and the ability to be en-zymatically amplified. It may be that flexible RNA structures provide anadvantage during the in vitro selection, and that extremely stable struc-tures are selectively depleted from the pools due to poor amplification.The selection pressures in vitro and in vivo are obviously distinct.

The generation of aptamer sequences naturally results in a phylogenetic-like family of sequences. Some nucleotides, as shown in Figure 1a–c, arecompletely conserved in every individual aptamer clone that is identified.Some nucleotides are quite variable, and their identity is unimportant, buttheir presence is required for function. These nucleotides are often in-volved in stacking or packing interactions, or as structural linkers. In allof the four ligand-RNA complexes, the conserved nucleotides in the RNAsequence were either directly involved in formation of the unique RNAarchitecture of the binding site, or were directly involved in contacts to theligand. Thus, the structure of these RNAs provides a rationale for thefunctional role of the conserved nucleotides.




DRUG–RNA INTERACTIONS: AMINOGLYCOSIDES

Paromomycin-A-site Ribosomal RNA Complex

Aminoglycoside antibiotics bind to the ribosome and inhibit translation(Davies et al. 1965). Beyond the obvious pharmacological importance ofthese compounds, they have provided fundamental insights into the mech-anism of protein synthesis. Aminoglycosides bind to 16S ribosomal RNAin the 30S ribosomal subunit (Moazed and Noller 1987) and cause mis-reading of the genetic code. A large body of biochemical and genetic datasupports a highly conserved binding site in ribosomal RNA that is near theaminoacyl-tRNA-binding site (A site). RNA oligonucleotides (Fig. 4c) thatcorrespond to this region of 16S rRNA (Purohit and Stern 1994; Recht etal. 1996) bind aminoglycosides in the same manner as the ribosome.

Aminoglycoside antibiotics contain common chemical features thatare required for drug action (Fig. 4a,b). All active aminoglycosides con-tain a nonsugar deoxystreptamine ring (ring II), and a sugar aminoglucosering (ring I) is always attached at position 4 of ring II, although the sub-stitution pattern of this ring can vary. The number of additional rings andposition of attachment to ring II vary as well. The 4,5 disubstituted com-pounds have an additional 5-membered sugar ring attached at position 5of ring II. Neomycin and paromomycin are members of this class thathave a fourth ring attached to the ribose sugar. The 4,6 disubstituted com-pounds have an additional 6-membered sugar ring attached at position 6of ring II. The aminoglycosides are positively charged at biological pH,with charges of +2 to +5, yet there is no strong correlation between an-tibiotic activity and total charge.

Figure 4 Aminoglycoside antibiotics and their RNA-binding sites. (a) Paro-momycin; (b) tobramycin; (c) E. coli 16S ribosomal RNA A-site oligonucleotide;(d ) tobramycin aptamer. Nucleotides involved in ligand contacts are shown ingreen.




High-resolution structures of aminoglycoside-rRNA complexes(Fourmy et al. 1996, 1998a,b) provide insights into how aminoglycosideantibiotics recognize ribosomal RNA and how they might interfere withtranslation. As suggested by biochemical data, the antibiotic binds in theRNA major groove, in the asymmetric internal loop (Fig. 5a). The struc-ture of the complex in the region of the internal loop is well defined by theNMR data, as are rings I and II of paromomycin, whereas rings III and IVare more disordered. Paromomycin adopts a specific L-shaped structure,with rings II, III, and IV in a line along the major groove, and ring I posi-tioned approximately 90˚ from the other three rings. Chemical groups thatare common among all aminoglycosides make specific hydrogen bonds tobases and phosphates in the major groove.

Distortions in RNA structure allow formation of the antibiotic bindingsite. Two noncanonical base pairs are formed in the asymmetric internalloop, resulting in a closed, stable structure for this loop. A U1406-U1495pair, which involves two hydrogen bonds, is formed. The asymmetric loopis closed by an A1408-A1493 base pair, with the Watson-Crick face ofA1408 contacting the N7, N6 face of A1493. The conformation of thisbase pair is buckled, and long N-N distances suggest water-mediated

Figure 5 Three-dimensional structures of aminoglycoside-RNA complexessolved by NMR spectroscopy. (a) Paromomycin-A-site RNA complex; (b) to-bramycin–RNA aptamer complex. The same coloring scheme as in Fig. 4 is used.




hydrogen bonding. A1492 is stacked below A1493 and is displaced to-ward the minor groove. The phosphodiester backbone is distorted at thejunction of the asymmetric loop (A1492/A1493) and the lower helicalstem (G1491). Ring I of paromomycin is located within a binding pocketformed by this distorted RNA structure. Mutations that disrupt the shapeof the binding pocket lead to reduced affinity for aminoglycosides anddrug resistance (Recht et al. 1996).

The structure of the A-site RNA–paromomycin complex explainsmany previous observations of aminoglycoside activity. Resistance en-zymes modify aminoglycosides on rings I and II (Shaw et al. 1993); theseenzymes include acetyl, phospho, and adenylyl transferases, and such modi-fications introduce electrostatic and steric penalties to aminoglycoside–RNA interaction. Interestingly, the 1-amino group of ring II, which makesa critical hydrogen bond to U1495, is not a target of resistance acetylases,as the acetyl group can readily exit from the top of the major groove,above U1495, with little steric penalty. Furthermore, semisyntheticaminoglycosides, such as amikacin, contain modifications at position 1that can also exit the major groove.

The A-site RNA undergoes only a minor conformational change uponbinding of aminoglycoside antibiotics (Fourmy et al. 1998a). In the freeRNA, the noncanonical base pairs in the asymmetric internal loop areformed, although there is evidence for conformational dynamics in theloop. Upon drug binding, the major conformational change occurs atA1492 and A1493, whose base moieties are displaced by 2–3 Å towardthe minor groove by ring I of paromomycin. A1492 and A1493 are uni-versally conserved nucleotides that have been implicated in the interac-tion of the ribosome with the tRNA anticodon–mRNA codon pair. The ef-fect of the aminoglycoside binding on their conformation may be relatedto aminoglycoside-induced misreading of mRNA codons.

Tobramycin–RNA Aptamer Complex

Aptamers that bind tightly to aminoglycosides have been selected (Lato etal. 1995; Wallis et al. 1995; Wang and Rando 1995) (Fig. 4d), and thestructure of a tobramycin–RNA aptamer complex has been solved (Jianget al. 1997). As with the paromomycin–ribosomal RNA complex, to-bramycin interacts with the aptamer in the major groove at the junctionbetween helical stem and nonhelical regions (Fig. 5b). Ring II of tobra-mycin interacts with a 5�GU3� helical step, as it does in the aminoglycoside–rRNA complexes. In contrast with the interaction with ribosomal RNA,tobramycin ring I is not in contact with RNA, but juts into solution. The




6�-NH2 on ring I was the site of derivatization of tobramycin for attach-ment to the column matrix, so that it can not contact the RNA as in theparomomycin–rRNA complex. Ring III is positioned relative to ring II inan almost parallel orientation near the G9-C18 and U10-A17 base pairsand makes close contact with the RNA. A residue in the hairpin loop, C15,is flipped out of the stacked hairpin and forms a lower flap on ring III. Theinteractions between C17 and ring III are apparently dipole-dipole andhydrophobic.

The high-affinity tobramycin–aptamer complex shows similar detailsof interaction as the aminoglycoside–ribosomal RNA complex. Ring II,the 2-deoxystreptamine ring, interacts along the edge of the major groove;in both structures a 6-membered sugar ring, whose identity is different inthe two interactions, makes more structure-specific recognition with theRNA within a binding pocket. In both structures, the bottom of the bind-ing pocket is an RNA base, although in the tobramycin structure, the ringinteracts edgewise with the RNA base, whereas in the paromomycin struc-ture, the sugar ring I is “flat” above the base. The difference in interactionof the aptamer and ribosomal RNA is that aminoglycosides must interferewith ribosomal function in addition to binding, whereas the aptamer wasselected solely on the basis of binding. This reflects the difference betweenhigh-affinity binding and functional binding of small ligands.

PEPTIDE–RNA INTERACTIONS

RNA-binding proteins in retroviruses and phages play a central role inregulation of replication (Karn et al. 1994). These RNA-binding proteinscontain a functional domain and an arginine-rich RNA-binding domain.Peptides that correspond to the arginine-rich domains of these proteins in-teract specifically with their RNA targets. Therefore, both the protein andRNA components in this system are modular. Despite the implication thatthe arginine-rich domain is a conserved structural motif, the NMR struc-tures described below reveal a variety of different folds and modes of in-teraction of these peptides with RNA.

HIV Tat–TAR Complex

The retroviral Tat proteins bind to an RNA stem-loop (TAR) at the 5� endof viral transcripts, and activate transcription (Frankel 1992). The Tat pro-teins are essential for viral replication and have been the focus of intensestudy. The Tat proteins contain a conserved, cysteine-rich core that is re-quired for transcriptional activation and an arginine-rich RNA-bindingdomain at the carboxyl-terminus. Peptides that correspond to the




arginine-rich domain bind to TAR RNA with affinity and specificitysimilar to those of the intact protein (Weeks et al. 1990). As discussedabove, free arginine also binds specifically to HIV TAR and drives a con-formational change in the RNA bulge. Peptide interactions with HIVTAR induce a similar RNA conformational change. The RNA-bindingdomain of Tat does not form a regular structure either in the absence ofRNA or when bound.

BIV Tat-TAR Complex

The biochemistry (Chen and Frankel 1994, 1995) and structure (Puglisi etal. 1995; Ye et al. 1995) of the bovine immunodeficiency virus Tat-TARinteraction have been delineated. BIV Tat protein has a domain structurerelated to that of HIV Tat, with sequence differences in the RNA-bindingdomain. Peptide fragments that correspond to the RNA-binding domainbind with high affinity to BIV TAR (Fig. 6b). Unlike HIV Tat protein, the

Figure 6 RNAs and arginine-rich peptides whose complexes are discussed inthe text. (a) HIV RRE; (b) BIV TAR; (c) phage λ boxB RNA; (d ) phage P22boxB RNA. Nucleotides involved in RNA–peptide contacts are highlighted ingreen. Lowercase nucleotides were changed from the biological sequence in theoligonucleotide construct. (e) Peptides corresponding to the arginine-rich RNA-binding motifs.




sequence of the arginine-rich domain is very important for specific recog-nition. Eight of 14 residues in the RNA-binding peptide cannot bechanged to other related amino acids without a significant loss of affinity.The sequence and secondary structure of BIV TAR RNA is distinct fromthat of HIV TAR. BIV TAR RNA contains two single uracil bulges, sep-arated by a single G-C base pair. The nucleotide requirements for Tatpeptide binding and function have been mapped and are clustered in thehelical stems near the uracil bulges.

Two structures of BIV Tat peptide bound to TAR RNA have beensolved by NMR, and the structures are essentially similar (Puglisi et al.1995; Ye et al. 1995). BIV Tat peptide is unstructured in the absence ofRNA, and adopts a β-strand conformation upon interaction with BIVTAR RNA. The peptide lies in the major groove of BIV TAR, near theuracil bulges (Fig. 7b). These two bulged nucleotides have different rolesin peptide interaction. U12 is extruded from the RNA helix and is disor-dered on the minor groove side of the helix. In fact, this bulge can bedeleted with little effect on binding affinity. In contrast, U10 interactswith an isoleucine side chain in the major groove, and this interaction be-tween a hydrophobic side chain and bulged nucleotide is required for spe-cific RNA–peptide interaction. In both structures, U10 is positioned nearan A-U pair in the major groove, and there is some evidence for a basetriple at low temperature, as observed in the HIV TAR–arginine complex;however, in the BIV peptide interaction, this triple is not required forbinding. The RNA undergoes a minor conformational change upon bind-ing, and the peptide recognizes the distortions in helical structure near theU10 bulge. In contrast to the HIV TAR–peptide interaction, there are mul-tiple specific amino acid–RNA contacts that guide complex formation.

The RNA-binding domain of BIV Tat contains three arginines, ofwhich two make well-defined contacts with helical guanosines. Theguanidinium groups of these arginines interact with the guanosine O6 andN7 groups, as discussed above for the arginine–HIV TAR interaction. Thethird arginine was less well defined in the structures, but makes essentialelectrostatic contacts in the major groove. A threonine side chain formshydrogen bonds with the phosphate oxygen of a helical base pair. TheBIV peptide contains three essential glycine residues. One is required forβ-turn formation, and the other two allow deep penetration of the peptideinto the relatively narrow major groove of the RNA helix: The lack ofside-chain steric bulk allows deep penetration. One glycine makes a main-chain hydrogen bond to a guanosine N7. The variety of specific RNA–peptide contacts may explain the relatively high specificity of this inter-action compared to other RNA-protein interactions.




Figure 7 Three-dimensional structures of RNA–peptide complexes. (a) HIVRev-RRE complex; (b) BIV Tat-TAR complex; (c) phage λ N-boxB–RNA com-plex; (d) phage P22 N-boxB RNA. RNA is blue and peptide is red.




Rev–RRE Complex

The HIV Rev protein is a small basic protein containing an arginine-richmotif that binds to the RNA regulatory sequence called the Rev responseelement (RRE). Binding of Rev to RRE results in a change of the relativeratios of unspliced and singly spliced to completely spliced mRNAs thatare exported from the nucleus to the cytoplasm, thus altering the patternof viral gene expression (Fritz and Green 1996). The Rev protein bears anuclear export signal as the effector domain and the basic region is both anuclear localization signal and the RNA-binding domain. Short peptidesfrom the basic region of Rev bind specifically to the RRE. The full RREis a large, complex structure that binds multiple Rev molecules, but a high-affinity Rev-binding site has been identified that binds a single Revmolecule. The minimal RRE is shown in Figure 6a.

The structure of the high-affinity Rev-binding site in the RRE in com-plex with Rev peptide has been determined using NMR (Battiste et al.1996). A similar aptamer–Rev peptide complex structure has also beensolved (Ye et al. 1996). The peptide forms an α helix that binds in thewidened major groove of the RRE (Fig. 7a). The RRE forms a continuoushelical structure that contains a G-A and a G-G base pair at the center ofthe widened major groove (Battiste et al. 1994). Two single nucleotidesare bulged out of the helix, but the stacking in the helix is otherwise con-tinuous on both strands. The geometry of the G-G base pair induces a pro-nounced kink in the backbone of one strand of the RNA, and this kink re-sults in a widening of the major groove by 5 Å compared to a standardA-form helix. This widening permits the α helix of Rev to penetratedeeply into the otherwise restricted groove of the RNA.

The overall shape of the groove is determined by the purine-purinebase pairs, but the specific recognition is mediated by side-chain contactsto bases in the major groove and to the backbone. Three arginine residuescontact guanine bases in a manner very similar to that observed inDNA–protein complexes. An asparagine residue forms hydrogen bonds tothe N6 and the O6 of the G-A base pair. One arginine makes contacts toparticular phosphates on the backbone. Finally, a threonine residue makesan N-cap structure that stabilizes the amino terminus of the helical pep-tide and simultaneously contacts a backbone phosphate.

N-Protein–Box B Complexes

Bacteriophage lambda and P22 N-proteins are transcriptional antitermi-nators that bind to RNA hairpin loops (Box B) in their respective mRNAs(Das 1993). The N-proteins contain amino-terminal arginine-rich RNA-




binding domains (Fig. 6e), and peptides that correspond to the arginine-rich domain interact specifically with boxB RNA hairpin loops (Fig. 6c,d)(Tan and Frankel 1995; Cilley and Williamson 1997). NMR studies haveshown that N-proteins are unfolded, and that only the RNA-binding do-main folds into an α helix upon complex formation (Mogridge et al.1998). The remainder of the N-protein presumably folds upon interactionwith other proteins involved in transcriptional antitermination. Flexiblestructures are an apparent hallmark of both arginine-rich domains and theviral/phage proteins that contain them (Frankel and Smith 1998).

The detailed structure of the lambda N-peptide-boxB RNA interaction(Legault et al. 1998) confirms the general features of RNA recognitiondiscussed above. The peptide adopts a bent α helix structure (Su et al.1997) upon binding the major groove of the RNA stem-loop (Fig. 7c). The5-nucleotide GAAGA loop forms a GNRA tetraloop fold with a shearedG8-A12 pair and stacking of loop nucleotides. The 4th guanine of theloop, G11, is extruded from stacking, and this guanine is required forbinding of the nusA factor to the N-protein–RNA complex. Arginine andglutamine side chains likely form specific hydrogen bonds with thesheared G-A pair, and a required tryptophan side chain stacks on theend of the tetraloop to further stabilize the loop fold. Other arginine andlysine side chains make electrostatic interactions with phosphate groupsof helical nucleotides. No base-specific hydrogen bonds are formed tohelical nucleotides, but a network of hydrophobic and electrostatic inter-actions, plus the specific bend of the α helix, lead to intimate RNA-protein recognition.

The structure of the P22 N-peptide-boxB RNA complex (Cai et al.1998) is similar to the λ N-peptide complex. The peptide forms an α he-lix, which interacts with a GNRA-type tetraloop and the major groove ofthe helical duplex (Fig. 7d). The α helix has a slight bend, which allowsmore intimate interaction with RNA. Basic amino acids, which are re-quired for specific complex formation, contact the phosphodiester back-bone of the RNA helix. No base-specific contacts are observed to helicalnucleotides. The boxB loop consists of 5 nucleotides, 4 of which (G9,A10, A12, and A13) form a -GNRA- fold with a sheared G9-A13 basepair. C11 is looped out and makes extensive hydrophobic contacts withpeptide side chains (alanine and isoleucine). Base-specific hydrogenbonds are formed between a critical arginine (Arg 6) and the O6 and N7positions of G9 in the tetraloop. The sheared G-A base-pair geometryleaves these positions available for protein recognition. The structureexplains the limited biochemical data on the interaction on P22 N-protein–RNA interaction.




Structural Variety of Arginine-rich Peptides

The striking result of these studies of arginine-rich peptides interactingwith RNA is their structural diversity. All arginine-rich peptides interactin the major groove of an RNA target, all are highly positively charged,and all are apparently unfolded in the absence of RNA. Upon RNA inter-action, the peptides form very different secondary structure folds toachieve recognition: an extended structure in HIV Tat, a β hairpin in BIVTat, and α helices in Rev and N-peptides. The different protein scaffoldsfor RNA recognition each achieve different detailed modes of recogni-tion. The β hairpin of BIV Tat is able to penetrate deep into the majorgroove, which is only slightly distorted, because it contains glycines,whereas α-helical peptides in Rev-RRE interact with a major groove thatis widened by purine-purine pairs. The structural diversity of arginine-richmotifs is a reflection of RNA structural diversity.

RNA STRUCTURAL PRINCIPLES

RNA structures and protein structures are both assembled from a combi-nation of secondary structures held together by tertiary interactions. How-ever, the stabilities of the secondary and tertiary interactions are very dif-ferent for the two classes of macromolecules. Protein secondary structurestend to be very weak on their own, and they are stabilized by strong ter-tiary packing forces. In contrast, the secondary structures of RNAs are ex-tremely stable in isolation, and the tertiary interactions are much weaker.Many RNAs are stabilized by binding of monovalent or divalent ions. Infact, the ligands can be an intimate part of the RNA fold, as in the AMPaptamer structure.

All of the structures discussed here, and in fact most other RNA struc-tures, contain large amounts of A-form helical secondary structures. Typ-ically, these very stable helices flank irregular structures that are usuallyless stable. Although the nonstandard regions of RNA structures providea varied context for ligand discrimination, it appears to be important tohold things together with A-form helices. Recognition by ligands oftenoccurs at the junctions between helices and nonstandard structures. Manyof the binding sites use the A-form helix as a platform on which the moreelaborate structures rest.

Base stacking is a defining element of the A-form helical segment,and it also appears that stacking is an important contribution to the stabi-lization of RNA tertiary structures. The amount of stacking of the basesappears to be maximized in the observed RNA structures. The exceptionsto this rule are bulged nucleotides that are completely unstacked, but in




these cases, there is usually a requirement for an unusual backbone ge-ometry that can only be accommodated by presence of a true bulge.

The global structures of the RNAs in these complexes are highlystacked. The planar nature of the bases apparently exerts a profound in-fluence on the geometries that RNAs can adopt. Even the much largertRNA (Saenger 1984) and P4-P6 domain of the group I intron (Cate et al.1996) are formed from two stacked helical segments, as are most of theother RNAs and RNA–ligand complexes whose structures are known (seeChapter 11). Just as protein structures are compacted to form a hydropho-bic core, RNA structures seem to form compact structures by maximiza-tion of base-stacking interactions. This tends to result in RNA structuresthat are more linear and less globular than is typical for proteins.

Hydrogen-bonding interactions also appear to be optimized in theseRNA structures. Simple base-pairing by hydrogen bonding is a commonlyobserved interaction that stabilizes both the A-form and nonstandard re-gions of structure. Although hydrogen-bonding interactions tend to bedominated by the bases, many hydrogen bonds occur to the phosphatebackbone and to the ribose sugar. Although the bases are a more obviousdevice to provide specific hydrogen bonds for a particular structure, thesugar and backbone can also provide specific contacts or elements of sta-bilization when placed in an unusual geometry.

Other common features in these complexes are the prevalence ofpurines in the conserved regions and the prevalence of non-Watson-Crickinteractions. The purines offer two opportunities for increased interactionscompared to pyrimidines. First, they have a larger surface area availablefor stacking interactions that predominate in these structures. Second, theyhave two distinct edges that can form multiple hydrogen bonds, and thusthey are well suited to form a variety of non-Watson-Crick interactions.

Electrostatic interactions also play a large role in shaping RNA struc-ture. In the nonhelical parts of RNA structures, phosphates may be broughtinto close proximity. This unfavorable effect may be offset by other favor-able interactions, such as ligand binding in the case of the aptamers, or bybinding of mono- or divalent ions. Magnesium ions are required for bind-ing of ligands to the FMN and theophylline aptamers, but not for the ATPaptamer or the HIV TAR-argininamide complex. In peptide and amino-glycoside–RNA complexes, Mg++ is not required for complex formation,reflecting the high positive charge density of these ligands.

LIGAND RECOGNITION

Ligand recognition of RNA makes use of the above structural principles.Ligand–RNA stacking plays a major stabilizing role in these complexes.




Theophylline, ATP, and FMN all contain a polycyclic aromatic ring,whereas argininamide bears a planar guanidinium group. These planargroups naturally lend themselves to favorable stacking interactions withthe planar aromatic bases present in RNA, and it is perhaps no surprisethat stacking is a prevalent feature in these RNA–ligand complexes. Theseligands use the free energy of stacking to drive fairly large conformationalrearrangements of the RNA upon binding. Stacking interactions are alsoused by the Rev and N-peptides, which contain aromatic side chains thatstack on RNA bases.

Stacking does not appear to provide extensive specificity in RNA–ligand interaction. The aromatic rings on the aptamer ligands contain exo-cyclic chemical groups that can be recognized by hydrogen bonding. Thespecificity of the AMP–RNA and theophylline–RNA recognition arise inlarge part due to a collection of hydrogen bonds to the aromatic chemicalgroups.

Aliphatic ligands also pack tightly against the RNA. The argininegroups in the Tat–TAR interaction make hydrogen bonds to a G-C pair,but also pack against the uracil of the base triple. In the aminoglycoside–RNA complexes, the aminoglucose ring is packed directly above a G-Cpair. These interactions between polar aliphatic ligands and the RNAbases may be stabilized by dipolar contacts. Hydrophobic ligand–RNAinteraction has also been observed. In the BIV Tat-TAR complex, an iso-leucine side chain packs against a bulged uracil.

Electrostatics are a major contribution to RNA–ligand interaction.Many of the ligands discussed here are positively charged, and these li-gands (aminoglycosides and arginine-rich peptides) interact in the majorgroove of the RNA. Obviously, RNA is negatively charged, but thischarge density is focused in the major groove, where the phosphate oxy-gens point. Magnesium binding in RNA major grooves has been shown tostabilize unfavorable RNA–RNA interactions. Likewise, the binding ofpositively charged ligands in the major groove also stabilizes unstableRNA backbone conformations. The long-range effects of electrostatic in-teractions can also be a driving force for conformational changes.

Whereas the small aromatic and aliphatic ligands fit tightly into well-packed RNA-binding pockets, the larger ligands—aminoglycosides andpeptides—have more extended interaction surfaces with their targetRNAs. These ligands are all positively charged and bind in the majorgroove. Electrostatic and hydrogen-bonding contacts are made with anumber of base and backbone positions along the length of a helicalstretch. Specificity is achieved by specific hydrogen-bonding interactionswith RNA bases and by recognition of some structural distortion: an in-




ternal loop, bulge or hairpin loop. For bulky peptides to fit in the majorgroove, widening of the groove by these distortions is often required. Be-cause of the variety in their side-chain composition, the peptide can usean ensemble of electrostatic, hydrogen-bonding, hydrophobic, and stack-ing interactions to achieve recognition.

The large conformational changes observed in aptamer–RNA com-plexes are more subtle in biological RNA–ligand complexes. The grooverecognition in the aminoglycoside and peptide complexes leads to less re-arrangement than the stacking interactions of aromatic ligands. None-theless, these subtle manipulations of RNA structure may be critical tomodulating RNA function. In the case of aminoglycoside antibiotics, theminor conformational change observed on binding may be the origin ofantibiotic action.

CONCLUSIONS

Structural studies on RNA–ligand complexes have provided insights intohow ligands recognize RNA. The ligands utilize the same range of inter-actions that stabilize RNA itself: stacking, hydrogen bonding, and elec-trostatics. RNA is a hospitable site for ligand recognition, and RNAclearly can form binding pockets for small molecules. The basic chem-istry of enzymes requires small molecule substrate and cofactor binding.The small ligand–RNA complexes discussed here highlight how primi-tive RNA machines may have bound small-molecule substrates in theRNA World.

ACKNOWLEDGMENTS

The authors thank D. Patel and L. Kay for providing unpublished coordi-nates, M. Recht and S. Blanchard for critical reading of the manuscript,and the members of the Puglisi and Williamson groups for stimulatingdiscussions.

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