Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins

Advanced Review

Recognition modes of RNAtetraloops and tetraloop-likemotifs by RNA-binding proteinsRoopa Thapar,1,2,3∗ Andria P. Denmon3 and Edward P. Nikonowicz3

RNA hairpins are the most commonly occurring secondary structural elements inRNAs and serve as nucleation sites for RNA folding, RNA–RNA, and RNA–proteininteractions. RNA hairpins are frequently capped by tetraloops, and based onsequence similarity, three broad classes of RNA tetraloops have been defined:GNRA, UNCG, and CUYG. Other classes such as the UYUN tetraloop in histonemRNAs, the UGAA in 16S rRNA, the AUUA tetraloop from the MS2 bacteriophage,and the AGNN tetraloop that binds RNase III have also been characterized.The tetraloop structure is compact and is usually characterized by a pairedinteraction between the first and fourth nucleotides. The two unpaired nucleotidesin the loop are usually involved in base-stacking or base-phosphate hydrogenbonding interactions. Several structures of RNA tetraloops, free and complexed toother RNAs or proteins, are now available and these studies have increased ourunderstanding of the diverse mechanisms by which this motif is recognized. RNAtetraloops can mediate RNA–RNA contacts via the tetraloop–receptor motif, kissinghairpin loops, A-minor interactions, and pseudoknots. While these RNA–RNAinteractions are fairly well understood, how RNA-binding proteins recognize RNAtetraloops and tetraloop-like motifs remains unclear. In this review, we summarizethe structures of RNA tetraloop–protein complexes and the general themes thathave emerged on sequence- and structure-specific recognition of RNA tetraloops.We highlight how proteins achieve molecular recognition of this nucleic acidmotif, the structural adaptations observed in the tetraloop to accommodate theprotein-binding partner, and the role of dynamics in recognition. © 2013 John Wiley& Sons, Ltd.

How to cite this article:WIREs RNA 2013. doi: 10.1002/wrna.1196

∗Correspondence to: [email protected] Department of Structural Biology, Hauptman-Woodward MedicalResearch Institute, Buffalo, NY, USA2 Department of Structural Biology, SUNY at Buffalo, Buffalo, NY,USA3 Department of Biochemistry and Cell Biology, Rice University,Houston, TX, USA

Conflict of interest: The authors declare no competing financialinterest.This article was published online on October 3, 2013. An errorin Figure 6 was subsequently identified. This notice is included toindicate the error has been corrected on October 16, 2013.

OVERVIEW

The RNA stem-loop or hairpin is the most commonsecondary structure motif in RNA.1 It was first

described in 1983 by Woese et al. to exist ineubacterial 16S-like ribosomal RNA (rRNA).2 Theloop motif that caps the A-form RNA helix isstructurally important to initiate RNA folding3 asit allows the phosphodiester backbone of single-stranded RNA to fold back on itself, therebyfacilitating formation of an intramolecular double-stranded RNA helix. Approximately 55% of RNAhelices in Escherichia coli 16S rRNA and 38% of RNAhelices in 23S rRNA are capped by tetraloops.1,2,4

Several families of RNA tetraloops have been classified

© 2013 John Wiley & Sons, Ltd.

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based on phylogenetic analysis, the geometry of thebackbone, and the interactions between the loopbases. These are the GNRA,1,2 UNCG,5 CUYG,1

GANC,6 (A/U)GNN,7,8 and UUUM9,10 tetraloops(where G is guanine, A is adenine, U is uracil, Nis any base, R is a purine, Y is a pyrimidine, and Mis either adenine or cytidine). The GNRA and UNCGtetraloops account for >70% of all tetraloops foundin rRNA.2

RNA tetraloops generally form compact andstable structures. The thermodynamic stability ofRNA hairpins depends on the size and sequenceof loop nucleotides. In general, tetraloops aremore stable compared with smaller or larger loopscontaining the same stem because they are ableto effectively minimize unfavorable base–solventinteractions via base-stacking, base-phosphate, andbase-ribose hydrogen bonds.11 Among all tetraloops,the UNCG, GNRA, and CUUG families are the moststable. The stability of these classes is attributed tobase pairing between the nucleotides at the 1 and 4positions, base-stacking, and 2′ OH-base hydrogenbonds in the loop.11

Hairpin loops participate in tertiary interactionsin large RNAs such as rRNAs12–14 and self-splicingRNAs15 that allow these molecules to fold andfunction. A well-characterized example is the P4–P6domain of the group I intron.15 The crystal structure ofthis motif shows a network of stabilizing base-stackingand 2′ OH hydrogen bonding interactions between aGAAA tetraloop and the receptor nucleotides locatedin a distal helix. The RNA kissing-loop motif ischaracterized by Watson–Crick (W-C) base pairingbetween loop nucleotides of two different hairpinloops, as observed in HIV-1 genomic RNA.16 RNApseudoknots, formed by W-C base pairing of the loopnucleotides with single-stranded RNA adjacent to theloop, are diverse and found in ribozymes,17 telom-erase RNA,18 and self-splicing introns.19 While theseRNA–RNA interactions involving RNA tetraloops arefairly well understood, little is known about the mech-anisms by which RNA tetraloops recognize proteins.

RNA tetraloops also function as recognitionsites for proteins in ribonucleoprotein complexes. Thewidely held view is that solvent exposure of loopnucleotides could play a role in sequence-specificmolecular recognition particularly via ‘induced-fit’mechanisms that are associated with RNA–proteincomplexes.20–22 Solution nuclear magnetic resonance(NMR) structures have been reported for the retroviralRous sarcoma virus (RSV) nucleocapsid (NC) proteinbound to a UGCG tetraloop,23 bacteriophage N-peptide-boxB GNRA-like tetraloop complexes,24–26

the Staufen dsRBD-UUCG tetraloop complex,27 and

the Rnt1p RNase III dsRBD bound to AGNNcognate and noncognate sequences.28,29 X-ray crystalstructures are available for the bacteriophage MS2coat protein bound to hairpin tetraloops,30–33 therestrictocin endonuclease bound to the GNRAsarcin/ricin loop (SRL) of 23S/28S rRNA,34 andthe SLBP RNA-binding domain/ERI1/histone mRNAstem-loop ternary complex.35 These structural studiesallow us to gain insight into the versatile modes ofhairpin loop recognition.

In this review, we examine our currentunderstanding of how specific recognition of RNAtetraloops and RNA tetraloop-like motifs is mediatedby RNA-binding proteins. Although general reviewson RNA hairpin structure, RNA–protein interactions,and RNA–RNA interactions are available, themolecular mechanism(s) by which RNA tetraloopsrecognize their protein targets has not been reviewedyet. RNA hairpin structure,11,14,36 RNA–proteininteractions,37,38 and RNA–RNA interactions12,14,39

have been reviewed and are not discussed in thisreport. High-resolution structures of free and boundRNA tetraloops are now available from severalsubfamilies. The existing structural, thermodynamic,and kinetic data allow us to scrutinize the differentmodes of tetraloop–protein recognition in detail andemphasize the various themes that have emerged.

SPECIFIC BASE RECOGNITION BY AHYDROPHOBIC POCKET

The Retroviral RSV NC ProteinUNCG-Type SL3 ψ-RNA ComplexThe UNCG family of RNA tetraloops forms unusuallystable hairpins.40,41 Several NMR and crystalstructures of UUCG41–44 and UACG45 containingRNA hairpins exist. The UUCG tetraloop in particular(Figure 1(a)) has been characterized by both NMRand X-ray methods43,44 and is essentially a biloop.It is distinguished by a bifurcated hydrogen bondbetween the first U carbonyl and the imino and aminoprotons of the fourth G (Figure 1(a)). The fourth Gadopts a syn configuration about the glycosidic bondthat is required for the stability of this tetraloop. Thestructure of the UUCG tetraloop is further stabilizedby a base-stacking interaction between the C and thefirst U as well as a base-phosphate hydrogen bondbetween the C amino proton and the U–U bridgingphosphate. The backbone turns between the first andsecond nucleotides. These features are conserved in allcharacterized UNCG tetraloops.

The RSV NC protein binds the UGCG tetraloopof stem-loop SL-C from the RSV Mψ packaging


WIREs RNA Recognition modes of RNA tetraloops and tetraloop-like motifs

(a) (b)

(c) (d)

FIGURE 1 | Structure of the N-terminal zinc knuckle ofthe Rous sarcoma virus (RSV) nucleocapsid (NC) proteinbound to the UGCG tetraloop of stem-loop SL-C from theRSV Mψ packaging signal (PDB code 2IHX). (a) Summaryschematic showing the hydrogen bonding and stackinginteractions observed in the free UUCG tetraloop of the P1helix from group I self-splicing introns (PDB code 1HLX).(b) Summary schematic showing the hydrogen bondingand stacking interactions observed in the tetraloop whenbound to the zinc knuckle (PDB code 2IHX). The sugarpuckers and base configurations are color coded accordingto the key shown below the schematic. (c) The zincknuckle is shown in purple and the RNA is in green. Theside chains of Tyr22 and Tyr30 stack against the guaninenucleotide G218 of the tetraloop. The configurations of theribose and bases correspond to the color code shown inthe summary schematic (b). (d) Surface representation ofthe RSV zinc knuckle showing the hydrophobic cleft thataccommodates G218.

signal,23 which corresponds to a 160-nucleotidefragment in the RSV 5′ untranslated region (5′ UTR).The RSV Mψ is sufficient to direct packaging of RNAsinto viral particles.46 An 82-nucleotide subfragmentof Mψ (termed μψ) is the minimal binding site forthe RSV NC protein.47 The predicted structure ofμψ consists of three stem-loops: SL-A, SL-B, and SL-C. An AUG initiation codon of open reading frame3 lies between SL-A and SL-B. Isothermal titrationcalorimetry (ITC) was used to show that the RSVNC protein interacts with μψ RNA with a 1:1stoichiometry and a Kd of 1.9 nM in 10 mM NaCl.The affinity of the RSV NC protein for the isolatedhairpin SL-C is 350 ± 60 nM, but the RSV NC proteinshows no appreciable binding toward hairpin SL-A orSL-B. A second binding site for the RSV NC proteinis the AUG initiation codon,48 and both the SL-Chairpin and the AUG site are required for formationof a high-affinity (Kd ∼1.9 nM) μψ RNA–NC proteincomplex.48 Intriguingly, replacement of the UGCGtetraloop in SL-C by a structurally stable GAGAtetraloop in the context of the intact μψ RNAdecreases binding to the RSV NC by 1500-fold,yielding a Kd of 2.8 ± 0.7 μM.48 Substitution of theSL-A and SL-B loops by GAGA has no effect on RNAaffinity for NC. Replacement of the UGCG tetraloopby a GAGA tetraloop also reduces viral infectivity,suggesting that reduced binding of RSV NC to theSL-C GAGA tetraloop affects viral assembly in vivo.48

The RSV NC protein binds the U217GCGtetraloop of SL-C via a CCHC-type zinc knucklemotif (Figure 1(c) and (d)). Solution NMR revealsthat the specific interaction between the N-terminalzinc knuckle and the U217GCG tetraloop23 is centeredon G218 (Figure 1(b)–(d)). Based on NMR and X-ray crystal structures of related UUCG and UACGtetraloops42–45 and the lack of observed NOEsbetween G218 and the RNA hairpin in the RSVNC/SL-C complex, it is inferred that G218 is likely tobe solvent exposed in the free RNA. When bound tothe RSV NC protein, G218 is completely buried in ahydrophobic pocket in the zinc knuckle (Figure 1(c)and (d)). The base of G218 forms π–π interactionswith the aromatic side chains of Tyr-22 and Tyr-30in a hydrophobic pocket defined by the side chains ofTyr-22, Tyr-30, Leu-20, and Gln-31 (Figure 1(b)–(d)).The G218 base also makes four hydrogen bonds withthe main chain of the zinc knuckle (G218 O6/Tyr-22NH, G218 O6/Gln-31 NH, G218 N1H/Leu-20 CO,and G218 NH21/Leu-20 CO). The network of hydro-gen bonding and stacking interactions mediated by theguanine base of G218 indicates that this nucleotideplays a key role in specific recognition of SL-C bythe hydrophobic pocket of the RSC NC protein. Inaddition to G218, the base of G220 packs against theside chains of Tyr-30 and Ala-17, and the ribose ofC216 packs against the side chain of Ala-32 (Figure 1).A similar mode of nucleotide–protein interaction has



been observed in the structure of the HIV-1 NC pro-tein bound to the SL3 ψ-RNA that has an unrelatedGGAG tetraloop.49 The solvent-exposed tetraloopguanine bases at the second and fourth positions ofthe GGAG loop are buried in separate hydrophobicpockets in the two zinc knuckles of the HIV-1 NCand stack against either Phe or Trp side chains.

Interestingly, the backbone and sugar confor-mations of the RSV SL-C Mψ-RNA tetraloop in thecomplex mirror free UNCG structures (Figure 1(a)and (b)) and neither the U217GCG SL-C tetraloop northe zinc knuckle motif appears to undergo a majorconformational change upon binding. Although thefree structure of the UGCG tetraloop is not available,it is possible to infer the potential free structure ofthis tetraloop based on structures of other tetraloopsfrom this family, and this structure concords with theNMR data for the UGCG tetraloop in the complex. Inthe RSV NC/SL-C complex, U217 and G220 are basepaired via interactions between the U217 2′ OH andthe G220 O6 as well as between the U217 O2 andG220 NH and NH2, as expected for the free RNA.The U217 O4 and G220 NH and NH2 also interactvia a bifurcated hydrogen bond. The phosphate 5′ ofG218 forms a hydrogen bond with the C219 NH2 andthe glycosidic bond of G220 is in a syn configuration.The U217 base stacks upon the closing C216•G221base pair of the stem and C219 stacks upon theU217•G220 base pair. These hydrogen bonding andstacking interactions are identical to those observed instructures of the free UNCG RNA tetraloops. There-fore, the structure of the RSV NC protein boundto the UGCG tetraloop of stem-loop SL-C suggeststhat the RNA does not undergo a conformationalchange in the complex and sequence-specific recog-nition of the UNCG loop may occur primarily viathe variable second position that is solvent exposed.In contrast, the three conserved nucleotides in thetetraloop are likely important to stabilize the loopstructure.

ADAPTIVE BINDING OF A HELIX INTHE MAJOR GROOVE OF A GNRAFOLD

The λ and P22 Bacteriophage N-Peptides inComplex with GNRA-Like boxB RNAsThe GNRA family of RNA tetraloops is widelydistributed in rRNAs,1,50 ribozymes,51 phageRNAs,24–26 the signal recognition particle,52,53 andviral internal ribosomal entry sites.54 All eight familymembers of this subgroup have a very similar back-bone conformation and are distinguished by a network

of hydrogen bonding and stacking interactions.15,55–57

There are several structures available for GNRAtetraloops.51,55,56 Analogous to the UNCG tetraloop,these sequence motifs form biloops where the G andA form a sheared G-A base pair (Figure 2(a)). Insuch a base pair, the G NH2 forms a hydrogen bondwith the phosphoryl oxygen 5′ of the A and the G2′ OH forms a hydrogen bond with the N7 of thepurine at the third position. The turn of the phosphatebackbone between the first and second nucleotides issimilar to that of the U-turn motif58 and the last threebases sequentially stack on each other on the 3′ sideof the stem. The second and third nucleotides can bedynamic and their sugar puckers frequently exchangebetween the C2′-endo and C3′-endo conformations.55

Notably, the functional groups of the last three basescan be an important readout for RNA–RNA andRNA–protein interactions.

It is important to recognize that the varioussubfamilies of tetraloops are structural ‘motifs’ thatare defined by their unique backbone geometry andinteractions between the loop bases. Kinetic andstructural data indicate that although the sequenceof the tetraloop imposes structural constraints,these motifs can occur within the context of largerpentaloops and hexaloops.59 Early kinetic studies onthe group II intron showed that insertion of one ortwo nucleotides into a GAAA tetraloop of the D5domain (to yield a GAXnAA type of pentaloop orhexaloop) did not dramatically affect the catalyticactivity of the group II intron ribozyme.59 Thissuggested that larger loops may form a GAAA-likestructure and participate in the tetraloop–receptormotif interaction. The NMR structure of the GAAGAboxB pentaloop bound to N-peptide24 later confirmedthat the boxB pentaloop adopts a GAAA fold thatsuperimposes on the GAAA tetraloop structure solvedby NMR56,60 as well as the crystal structures ofGAAA tetraloops from the hammerhead ribozyme61

and the P4-P6 domain of the group I intron15 withroot-mean-square deviation (RMSD) between 1.4and 1.6 A. The structure of a hairpin with a GCAUApentaloop from the U6 RNA (PDB code 1LC6) hasalso been shown to adopt a GNRA-like fold thatsuperimposes on a GCAA tetraloop (PDB code 1ZIH)with an RMSD of 2.4 A.62 The uridine at the fourthposition is extruded to solvent and is not part of theGCAA-like fold (Figure 2(b)).

The mode of N-peptide recognition bythe boxB GNRA-like hairpins present in thenascent-phage mRNA transcripts from lambdoidbacteriophages (λ and P22) is an example of molecularrecognition mediated by the GNRA-like familyof tetraloops24–26 (Figure 2(c)–(h)). The N-protein



(a) (b)

(c) (d) (e)

(h)(f) (g)

FIGURE 2 | Structures of λ and P22 bacteriophage N-peptides bound to the GNRA-like boxB RNA tetraloops. (a, b) The summary schematicshowing the hydrogen bonding and stacking interactions observed in the free GAAA tetraloop (PDB code 1ZIF) and free GCAUA GAAA-like tetraloop(PDB code 1LC6), respectively. (c–e) Structure of the λ N-peptide-boxB RNA complex (PDB coordinates are undeposited and were obtained from DrLegault, University of Montreal, Canada). The peptide is shown in purple and the RNA is in green. In (c), the summary schematic showing thehydrogen bonding and stacking interactions observed in the tetraloop when bound to the peptide is shown. The specific interaction of the bases withprotein side chains is highlighted. In (d), the shape-specific recognition of the bent helix of the λ N-peptide with the major groove of the RNA isshown. The RNA surface is shown in green. The small purple surface on the RNA shows the position of G11 in the tetraloop. The color codes aresimilar to that of the schematic shown in (c). In (e), the specific interactions between the protein side chains and the RNA bases are depicted incartoon format. (f–h) Structure of the P22-ARM peptide-boxB RNA complex (PDB code 1A4T). The peptide is shown in purple and the RNA is in green.The N-peptide forms a bent α-helix with extensive ionic interactions with the major groove face of the GNRA-like hairpin. The summary schematic isin (f), the surface representation in (g), and the side chain to base contacts depicted in cartoon format in (h).

plays an important role in regulating transcriptionelongation and transcription antitermination ofphage mRNA transcripts and is crucial for phagedevelopment.63,64 The 22-amino acid N-peptides formspecific complexes with their respective cis-actingboxB hairpins via arginine-rich motifs (ARMs).N-peptides bind their respective boxB RNAs withhigh affinity (Kd 5 nM)65,66 and have similar affinityfor the RNA as the full-length N-protein. Thereare three structures available for boxB/N-peptidecomplexes from the λ and P22 bacteriophages.24–26

NMR structures have been calculated for two relatedboxB hairpin/N-peptide complexes from λ-phage, aboxB hairpin with a GAAGA loop,24 and anotherwith a GAAAA loop.26 The NMR structures of bothcomplexes are very similar. Although the GAAGAand GAAAA loops are pentaloops, they adopt the

GNRA-like hairpin motif using nucleotides 1, 2, 3,and 5 and are identical to the GAAA tetraloop56,67

(Figure 2(a) and (c)). The fourth nucleotide ofthe pentaloop is flipped out in both structures.Replacement of the nucleotide at the fourth positionor deletion of the fourth nucleotide by any othernucleotide does not affect binding to the phageλN-peptide in electrophoretic mobility shift assay(EMSA).68,69 A Kd value similar to that of the wild-type hairpin/N-peptide interaction (5.2 nM) has beenreported for hairpins with substitutions at the fourthposition24,65 and a less than twofold decrease inbinding is observed for a fourth position deletionmutant in EMSA.68,69 In contrast, mutation of eitherthe G or the A of the sheared G-A base pairabolishes binding to the N-peptide,68,69 consistent



with the GNRA-like fold being essential for N-peptidebinding.

In the structure of the GAAGA λ-boxB hairpinloop complexed to a 22-amino acid arginine-rich λN-peptide (Figure 2(c)–(e)), the peptide forms a bentα-helix that packs against the major groove face onthe 5′ end of the RNA stem and three nucleotides (G1,A3, and A5) of the loop. A 120◦ bend between twohelical segments (Ala3-Arg10 and Arg12-Ala21) isstabilized by ionic interaction of Arg-11 with the loop-closing U•A base pair as well as hydrophobic contactsbetween the side chain of Gln-15 and the secondand third adenine bases in the loop24 (Figure 2(e)).The peptide undergoes a ‘disorder-to-order’ foldingtransition upon RNA binding to form a surface thatis complementary to that of the RNA hairpin. Thebent helix conformation is unique to the λ and P22N-peptide/boxB complexes and is not observed inother arginine-rich peptide–RNA complexes such asHIV-1 Rev70,71 or BIV-1 Tat.72,73 Formation of thecomplex is electrostatically driven and a cluster of fivearginines and two lysines form a positively chargedα-helical surface that interacts extensively with thenegatively charged phosphodiester backbone of theRNA (Figure 2(c) and (e)).

Hydrophobic interactions are also important forbinding. The aliphatic side chains of Arg-7 and Arg-8 contact the ribose as well as base (H5 and H6)protons of the C6 and U7 bases in the stem and thethird nucleotide (A10) in the loop. The methyl groupof Ala-3 packs against two cytidines in the stem. Mostimportantly, A9, which corresponds to the variable‘N’ position in the GNRA motif, stacks with theindole side chain of Trp-18 (Figure 2(c) and (e)).This interaction is important for specific recognitionof the λ boxB RNA by λN-peptide because otherphage N-peptides that are arginine rich but do nothave a Trp at this position are not able to form aspecific complex with the λ boxB RNA. It is alsocrucial for antitermination.60,67 Ultrafast dynamicsmeasurements using fluorescence show that the Trp-18/A9 stacking interaction regulates the conformationof the N-terminal end of the α-helix and is requiredfor proper assembly of peptide λ in the complexwith boxB RNA.74 Therefore, specific recognitionof boxB RNA by the λN-peptide is due to shapeand charge complementarity of the protein α-helixand the RNA major groove as well as the stackingof Trp-18 with the second adenine base of theGAA(G)A loop.

The NMR structure of the phage P22 N-peptidein complex with P22 boxB RNA25 suggests a similaradaptive mode of recognition by the P22 ARM (Figure2(f)–(h)). The N-peptide forms a bent α-helix with

extensive ionic interactions with the major grooveface of the GNRA-like hairpin (Figure 2(g)). The P22boxB pentaloop has the sequence GACAA and in thestructure of the complex with the P22 N-peptide itis the C that is extruded from the GNRA-like fold(Figure 2(f)). This cytidine is involved in extensivehydrophobic contacts with the methyl and methylenegroups of Arg-10 and Arg-11, the side chain of Ile-15,and the imidazole ring of His-7 (Figure 2(f)).

The broad specificity of the GNRA-like fold forARMs is largely determined by shape and electrostaticcomplementarity of the GNRA-like loop for arginine-rich containing peptides. The intermolecular contactsobserved between loop nucleotides 1, 2, and 3with the ARM-containing peptides and extensiveionic interactions with the major groove face of theGNRA-like motif indicate that the GNRA fold isan important determinant of specificity for ARM-containing peptides. The 5′ strand of the RNA stem isinvolved in extensive ionic and van der Waals contactswith the peptide and these interactions are conservedin all structures. The more restricted specificity ofN-peptide-boxB recognition within ARM-containingproteins arises from differing interactions of the N-peptide with the GNRA-like loop. In the case of theλ phage, the stacking interaction between Trp-18 andA9 is critical. In P22, the bulged cytidine of the loopcontacts the aliphatic side chains of Arg-10, Arg-11,and Ile-15 residues and stacks against the side chainof His-7. In both the λ and P22 phage structures,the conserved nucleotides of the GNRA-like tetraloopmotif are important for the overall fold of the loopbut not important for specificity.

In summary, the boxB/N-peptide complexes arean example of ‘induced-fit’ or adaptive mode ofrecognition of an RNA hairpin by a bent α-helixin the major groove of a GNRA fold. The N-peptide isdisordered in the absence of the RNA and adapts to theshape of the major groove face of the GNRA-like fold,forming a well-defined helical structure. Although nostructure is available for the free GAAGA, GAAAA,and GACAA RNA pentaloops, the structure of the freeGCAUA pentaloop from the U6 RNA (Figure 2(b))shows a typical GNRA fold formed by the GCAAresidues with the extra uridine nucleotide flipped outand flexible in the context of the pentaloop. Whilethe GAAGA, GAAAA, and GACAA pentaloops havea backbone geometry that is similar to a GNRA-like fold when bound to the N-peptide, the extrudednucleotide in the RNA loop is rigid and ordered,suggesting that motion may be more restricted in theGNRA-like RNA loop when bound to the N-peptide.Therefore, conformational flexibility of both the RNAand the N-peptide may be necessary to exploit surface



complementarity in the major groove of the GNRA-like RNA loop.

SHAPE-SPECIFIC RECOGNITION OFTHE (A/U)GNN TETRALOOP MINORGROOVE BY A HELIX

(A/U)GNN Cognate and NoncognateTetraloops in Complex with theEndonuclease Rnt1pThe double-stranded RNA-binding domain (dsRBD)is one of the most abundant structural domains foundin RNA-binding proteins.75 Canonical dsRBDs consistof 65–70 amino acids with a αβββα folding topology.dsRBDs are highly specific for dsRNA and do not binddsDNA or ssRNA. The specificity of dsRBDs towarddsRNA arises from extensive interaction of the proteinbackbone and side chains with the 2′ OH groups ofthe dsRNA in the minor groove.76 Structural studiesof dsRBD–RNA complexes reveal that in general,these domains bind RNA in a structure-specific butsequence-independent manner. Structures of RNA-bound dsRBDs are available for Xenopus laevisXlrbpa,76 Drosophila Staufen,27 TRBP2,77 ADAR2,78

Aquifex aeolicus RNase III,79 Arabidopsis HYL1,77

and Rnt1p.28,29,80 In all cases, helix α1, helix α2,and the β1–β2 loop mediate interactions with theconsecutive minor, major, and minor grooves on oneface of the RNA, respectively.

Rnt1p is an RNase III homolog from buddingyeast that has a Dicer-like endoribonuclease domainthat cleaves double-stranded RNA and is essentialfor RNA processing.81–84 Rnt1p is unique in that itis the only known RNase III that binds substratescontaining a (A/U)GNN tetraloop via its N-terminaldsRBD and cleaves the RNA 12–14 bp away from thetetraloop in a molecular-ruler type of mechanism.85,86

The (A/U)GNN tetraloops are commonly found inyeast small nuclear RNAs (snRNAs), small nucleolarRNAs (snoRNAs), and rRNAs that are all substratesfor Rnt1p.87 Most Rnt1p substrates have A and G asthe first two nucleotides. The only exceptions are U1snRNA that has a U instead of an A at the first positionand the AAGU tetraloop from the snoRNA, snR48.88

The (A/U)GNN RNA tetraloop–Rnt1p interaction isof high affinity (Kd 10–12 nM)85,86 and decreases100-fold upon deletion of the dsRBD, indicatingthat the dsRBD is important for target selection.86

In addition, replacing the AGGA tetraloop with aGUGA tetraloop decreases the efficiency of cleavagethat is directly attributed to a fourfold decrease inthe affinity of Rnt1p for the GUGA mutant.86 Thereare several structures now available for free Rnt1p

dsRBD,80,89 free (A/U)GNN RNA substrates,8,90 andcomplexes of Rnt1p with cognate28 and noncognate29

tetraloops. These structures along with informationabout backbone dynamics from NMR spin-relaxationmeasurements80 have provided detailed insight intothe mechanism of Rnt1p dsRBD–tetraloop RNArecognition.

Solution NMR studies8 of the free A6GAA andAGUU tetraloops (Figure 3(a)) reveal a well-orderedfold that is similar to the solution NMR structureof the unmethylated version of the UGAA tetraloopconserved at the 3′ end of eukaryotic 18S rRNA,7 butvery different from the UNCG and GNRA family oftetraloops. A characteristic feature of the (A/U)GNNfamily of tetraloops is the conserved G at thesecond position that adopts a syn configuration aboutthe glycosidic bond allowing the sugar-phosphatebackbone to reverse direction by 180◦ on the 3′ sideof the guanosine (Figure 3(a)). The sharp turn exposesthe nonbridging phosphate oxygen atoms between theG and the N nucleotides while the G base stacks onthe preceding adenine and the closing base pair of thestem. This loop conformation creates a binding pocketthat is recognized by Rnt1p. The syn configuration ofthe G and the sharp turn are strong determinantsof (A/U)GNN tetraloop structure and Rnt1p dsRBDrecognition. In addition, in the free RNA hairpin, thefirst A and the second G stack over the closing baseof the stem at the 5′ end, whereas the two adenines inthe third and fourth positions stack on the 3′ end ofthe closing base pair of the stem.

Solution NMR and X-ray crystal structures ofthe free89 and RNA-bound28 Rnt1p dsRBD reveal aconserved αβββα fold with an additional helix α3 thatis unique to Rnt1p (Figure 3(d)). In the free dsRBD,helix α3 is positioned near the C-terminus of α1, andpacks against the α1-β1 loop. The solution NMRstructure of the Rnt1p dsRBD complexed to a 14-bp hairpin with an AGAA tetraloop from the snR47snoRNA precursor has been solved28 (Figure 3(c)–(e)).Helix α1 of the Rnt1p dsRBD is the recognitionhelix and it binds the bend in the minor groove ofthe (A/U)GNN tetraloop by contacting the sugar-phosphate backbone, the ‘NN’ bases, and the top ofthe stem (Figure 3(c)). The N-terminus of helix α1 isextended by three residues in the RNA-bound complexand lies near the 5′ end of the tetraloop, whereasits C-terminus is near the 3′ end. Overall, helix α1undergoes a conformational change and adapts tothe shape of the RNA tetraloop in the minor groove(Figure 3(d) and (e)). Helix α1 bends at Ser-376 andundergoes an 18◦ rotation and translation toward theRNA.80 This conformational change is facilitated byhinge motion of the α1-β1 loop that also changes



(a) (b)

(c) (d) (e)

(h)(f) (g)

FIGURE 3 | Structure of the Rnt1p dsRBD bound to cognate (PDB code 1T4L) and noncognate (PDB code 2LBS) (A/U)GNN tetraloops. (a)Summary schematic showing the hydrogen bonding and stacking interactions observed in the free AGAA tetraloop (PDB code 1K4A) is shown. In (b),the summary schematic for hydrogen bonding and stacking interactions observed in the free AAGU tetraloop (PDB code 2HNS) is shown. (c) Summaryschematic showing the hydrogen bonding and stacking interactions observed in the complex formed by the cognate AGAA tetraloop bound to theRnt1p dsRBD (PDB code 1T4L). The sugar puckers and base configurations are color coded according to the key shown in Figure 1(c). Protein sidechains that contact the base and the sugars are depicted. (d) Overall structure of the Rnt1p dsRBD bound to the AGAA tetraloop. The protein is shownin purple and the RNA surface is shown in green. The protein contacts the successive minor, major, and minor groove via α-helix1, the β3-α2 loop,and the β1-β2 loop, respectively. (e) A close-up of the interaction of α-helix1 with the RNA tetraloop is shown. (f) Summary schematic showing thehydrogen bonding and stacking interactions observed in the noncognate AAGU tetraloop bound to the Rnt1p dsRBD complex (PDB code 2LBS). Proteinside chains that contact the base and the sugars are depicted. In panels (g) and (h), the specific contacts between the protein side chains of α-helix1and the RNA bases of the AGAA and AAGU tetraloops are compared. The AGAA tetraloop is in panel (g) and the AAGU tetraloop is in panel (h).

the orientation of helix α3. In helix α1, the side-chain guanidinium of Arg-372 and hydroxyl groupsof Ser-376 hydrogen bond to the 2′ OH of the ‘NN’nucleotides A17 and A18. The aliphatic portion ofthe Arg-372 side chain also makes van der Waalscontacts with the A17 and A18 bases (Figure 3(c)and (g)). In addition, residues Asp-367, Lys-371, andTyr-375 of helix α1 form contacts with the 2′ OH andribose of three base pairs in the A-form stem belowthe tetraloop. Furthermore, side chains from the N-terminus of helix α2 and the β3-α2 loop contact thesugar-phosphate backbone of the major groove ofthe A-form stem. Intriguingly, only the nonconservedbases are contacted by the Rnt1p dsRBD and thereare no contacts made to the conserved (A/U)G basesthat point into the major groove and are essential for

the general fold of the tetraloop. The RNA tetraloopdoes not appear to undergo a major conformationalchange to accommodate helix α1 in this complex.The only significant change in RNA structure is thata small bend in the backbone is observed at the topof the helix and below the tetraloop in response tobinding of the Rnt1p dsRBD. A superposition of thelowest energy conformers of the free AGAA tetraloopand the AGAA tetraloop bound to the Rnt1p dsRBDyields an RMSD of 0.99 A for backbone heavy atomsof the tetraloop and the closing base pair. Similar tothe structures of the free RNA hairpins containingAGAA,8 AGUU,8 and AGUC91 tetraloops, the RNAbackbone in the Rnt1p complex turns after the secondguanine that is in a syn configuration, the conserved Aand G bases stack on the 5′ end, and the nonconserved



bases stack on the 3′ side of the tetraloop. In summary,the structure of the Rnt1p dsRBD bound to theAGAA tetraloop shows that the dsRBD adapts tothe shape of the RNA tetraloop in the minor grooveand no sequence-specific contacts are made betweenthe protein and the RNA. The RNA undergoes only asmall conformational change upon protein binding.

In contrast to the cognate AGAA tetraloop,the structure of the Rnt1p dsRBD/noncognate AAGUtetraloop complex29 (Figure 3(f)–(h)) shows a moresignificant conformational rearrangement in theAAGU RNA tetraloop to accommodate the Rnt1phelix α1. The structures of the free noncognate(AAGU)90 and cognate (AGAA) tetraloops8 (Figure3(a) and (b)) are very different with an RMSD of 3.3 Afor sugar-phosphate backbone heavy atoms. The dif-ference between the tetraloop structures is particularlyimportant at the 3′ end of the loop. In the structure ofthe free noncognate AAGU RNA, the uracil is flippedout toward the solvent and does not form a base pairwith the adenosine at the 5′ end of the loop (Figure3(b)). The two adenosines on the 5′ side stack contin-uously with the closing base pair of the stem and thesugar-phosphate backbone turns between the adeno-sine and the guanine. Comparison of the structuresof the cognate Rnt1p dsRBD–AGAA and noncog-nate Rnt1p dsRBD-AAGU complexes shows that thebound states are remarkably similar. A superpositionof the lowest energy conformers of the Rnt1p-boundAAGU tetraloop29 and the AAGU tetraloop boundto the Rnt1p dsRBD28 complexes yields an RMSDof 0.97 A for backbone heavy atoms of the tetraloopand the closing base pair (not shown). When boundto the Rnt1p dsRBD, the nonconserved G17 and U18bases of the AAGU tetraloop undergo a large con-formational change to accommodate helix α1 (Figure3(b) and (f)). G17 and U18 point into the minorgroove and their backbones interact with the sidechains of Arg-372 and Ser-376 in helix α1 (Figure 3(f)and (h)). No interaction is observed between the firsttwo adenosines on the 5′ side of the tetraloop, sim-ilar to that observed in the cognate AGAA–dsRBDcomplex. The structural data on the cognateAGAA/dsRBD and noncognate AAGU/dsRBD com-plexes is supported by ITC measurements.29 Thetwo complexes have the same binding affin-ity (Kd = 2.23 ± 0.27 μM for the AAGU/dsRBD vs2.25 ± 0.15 μM for the AGAA/dsRBD) in 300 mMNaCl, but the AAGU/dsRBD undergoes larger changesin enthalpy (�H = −1.42 ± 0.06 × 104 kcal mol−1 forthe AAGU/dsRBD vs −0.92 ± 0.02 × 104 kcal mol−1

for the AGAA/dsRBD) and a significant decreasein entropy (�S = −21.8 kcal mol−1 K−1 for theAAGU/dsRBD vs −3.94 kcal mol−1 K−1 for the

AGAA/dsRBD) upon complex formation when com-pared with the AGAA/dsRBD complex. Therefore,the structure of the noncognate AAGU-capped hairpinbound to the Rnt1p dsRBD is an example of a cooper-ative conformational change in both the RNA and theprotein components to form a shape-specific complex.

The Rnt1p dsRBD–AGAA tetraloop complexis one of the few RNA–protein complexes wherethe contribution of motion and dynamics toRNA binding has been investigated.80 NMR-basedbackbone dynamics measurements for the free andRNA-bound Rnt1p dsRBD suggest that in generalthe dynamic changes in the protein correlate wellwith the conformational transitions that the proteinundergoes upon binding RNA. In the RNA-freestate, the Rnt1p dsRBD shows slow motions onthe microsecond–millisecond timescale (as measuredby the parameter Rex) for helix α1, the α1-β1loop, and helix α3. When bound to the AGAAtetraloop, the α1-β1 loop becomes more rigid, butresidues in helices α1 and α3 have larger Rex valuesindicating an increase in conformational exchange onthe microsecond–millisecond timescale. The increasein slow motions for these regions indicates that theRnt1p-AGAA tetraloop interface is not rigid and suchdynamics at the protein–RNA interface may reflecthow the dsRBD can adapt to different (cognate andnoncognate) RNA substrates.

COUPLED FOLDING AND BINDING OFA DISORDERED TETRALOOP

The MS2 AUUA Hairpin Loop RNAComplexThe MS2 coat protein binds a hairpin from its RNAgenome in a highly sequence- and structure-specificmanner.92–94 Interaction of the RNA hairpin withthe MS2 coat protein is the obligatory first step intranslational repression of the viral replicase gene.95

The free RNA hairpin consists of a seven bp stemwith a −7AUU−4A loop and an adenosine bulge(−10A) (Figure 4(a)). In the structure of the free RNA,the bulged −10A is intercalated between the flankingbase pairs of the stem. The −7AUU−4A tetraloop isflexible and multiple conformations are observed byNMR.96 The relaxation measurements suggest thatthe −6U, −5U, and −4A bases are very dynamic andpredisposed to reorganization, whereas −7A at thestart of the loop is stable.97

The MS2 coat protein is dimeric at lowconcentrations (up to 1 μM) but the dimers self-associate to form phage-like capsids at highconcentrations.98 The MS2 coat protein dimer binds



(a) (b) (c)

(d) (e)

FIGURE 4 | Structure of the MS2 coat protein bound to the AUUA tetraloop from its RNA genome. (a) Summary schematic showing the hydrogenbonding and stacking interactions observed in the AUUA tetraloop in the absence of the MS2 coat protein (PDB code 1D0T). (b) Summary schematicshowing the hydrogen bonding and stacking interactions observed in the AUUA tetraloop when bound to the MS2 coat protein (PDB code 1ZDH). Thesugar puckers and base configurations are color coded according to the key shown in Figure 1(c). Specific interaction of the bases with protein sidechains is depicted. (c) Overall structure of the MS2 dimer bound to the RNA hairpin. The protein is shown in purple cartoon and the RNA is shown ingreen. (d) Surface representation of the MS2 coat protein showing hydrophobic clefts that accommodate the −4A and −10A adenosines. The RNA isshown in green and the colors of the ribose sugar puckers correlate with the summary schematic in (b). The C3′-endo configuration is shown in blueand the C2′-endo configuration is shown in pink. (e) Close-up of the MS2–RNA-binding interface. The protein side chains are in gray and the RNAribose and bases are color coded according to the key shown in Figure 1(b).

the RNA hairpin with a Kd of 3.3 nM.99 Some insightinto the mechanism of RNA recognition by the MS2coat protein comes from stopped-flow fluorescenceexperiments.99 The kinetics measurements show abiphasic mode of interaction between the MS2 coatprotein and the RNA hairpin. The fast phase isdiffusion driven and occurs with a rate constantof 2 × 109 M−1 second−1. The salt dependence ofthe first phase suggests that it is dominated byelectrostatic steering effects. The slower phase appearsto correspond to the isomerization of the protein froma nonnative state to an RNA-binding competent stateprior to RNA binding.

Several crystal structures of MS2 bound to wild-type, mutant RNA hairpins, and RNA aptamers havebeen solved.31–33 The crystal structures of the MS2protein bound to the RNA hairpin reveal that theprotein is a symmetric dimer in which the RNArecognition face consists of a five-stranded β-sheetfrom each monomer98 (Figure 4(c)). The MS2 proteindimer binds asymmetrically to the RNA hairpinand contacts −4A and −10A directly (Figure 4(c)and (d)). The −4A base inserts into a hydrophobic

pocket formed by valine and lysine side chains inone monomer and the bulged −10A base insertsinto a hydrophobic pocket in the second monomer.Biochemical studies show that a pyrimidine is requiredat the −5 position in the loop. The U/C at the −5position stacks on the side chain of Tyr-85 of one ofthe MS2 monomers. The adenosine at the −7 positionin turn stacks with the −5U/C above it and the closingbase pair of the stem below. The sugars in the −4,−5, and −6 positions have C2′-endo conformationsand the bulged −10A has a C3′-endo conformation.Although the MS2 coat protein does not undergoa large conformational change upon RNA binding,the structure of the RNA tetraloop and bulge inthe complex is dramatically different from the NMRsolution structures96,97 of the same hairpin loop inthe absence of protein. In the free RNA, the sugars ofnucleotides in the −4, −5, and −6 positions have C2′-endo conformations, as observed in crystal structuresof the MS2–RNA complex; however the −5U and −4Abases are stacked on each other and the −7A and −6Ubases stack on each other. Therefore, the structuraldata suggest that the free RNA loop exchanges



between multiple states and either undergoes a largeconformational change upon binding MS2 or a minortetraloop conformer is selected upon binding.

RECOGNITION BY TETRALOOPUNFOLDING

Recognition of the GNRA SRL byRestrictocin

The Histone mRNA Hairpin-SLBP RBD-ERI1ComplexIn the examples discussed so far, several intriguingobservations have emerged. In many cases, thestructure of the free RNA tetraloop remains largelyunaltered when bound to the target protein.Second, there are several RNA–protein complexeswhere nonconserved bases in the tetraloop formRNA–protein contacts important for specificity whilethe conserved bases appear to be important forpreservation of the RNA tetraloop structure. Thereare, however, two examples that deviate from theseobservations wherein the RNA tetraloop undergoes alarge conformational change and becomes unfoldedupon binding the protein.

X-ray crystal structures of the SRL RNA boundto the α-sarcin ribotoxin restrictocin34 show that thehighly stable GNRA tetraloop is unfolded in the toxin-bound form (Figure 5(b) and (c)). The α-sarcin familyof ribonucleases is secreted by fungi100 and bindsthe SRL structure in 23S-28S rRNA. Restrictocinspecifically cleaves the SRL GAGA tetraloop betweenthe third and fourth loop nucleotides and impairsbinding of the elongation factors EF-G and EF-Tuto the ribosome, thereby stopping protein synthesis.The structure of the free SRL hairpin present in the50S ribosomal subunit consists of a typical GNRAtetraloop (Figure 5(a)) and a stem containing a bulged-G motif101 (Figure 5(d)). The bulged-G motif islocated 12 A from the cleavage site and is necessaryfor specific recognition by α-sarcin ribotoxins. Tominimize cleavage during crystallization, structuresof restrictocin were solved bound to RNA variantscontaining a 2′ O-methyl guanosine (mG) substitutionat the third position in the loop. This substitution isnot expected to alter the structure of the GNRAmotifs. In one variant, a 2′ deoxy adenosine (dA)was also introduced at the fourth position.34 In allstructures, the restrictocin contacts the major grooveface of the SRL RNA via three loops in the protein.Two lysine-rich loops make direct contacts with theguanine nucleotide and the backbone at the bulged-G (Figure 5(d)). The structure of restrictocin and the

bulged-G motif of the RNA hairpin remain unchangedwhen compared with the uncomplexed states.

Upon formation of the restrictocin–SRL RNAcomplex, the GAGA tetraloop shows base flipping,a change in the backbone conformation, andbase stacking that is characteristic of an unfoldedloop (Figure 5(a)–(c)). Two different unfoldedconformations are observed for the tetraloop in themG and dA SRL RNA substrates, indicating thattetraloop unfolding is not due to the methyl (mG)substitution. In each of these structures, the bases ofthe third G and fourth A flip out of the GNRA fold.As a result, the sheared G-A base pair characteristicof GNRA motifs is lost. In addition, the bases of thesecond, third, and fourth nucleotides no longer stack.The backbone also reverses direction between thesecond and third nucleotides in the restrictocin–SRLcomplex, in contrast to the free SRL, where thebackbone changes direction between the first andsecond nucleotides. The unfolding of the tetraloop ispresumably a result of protein binding. Superpositionof the restrictocin–SRL mG complex crystal structurewith the structure of the SRL in the 50S ribosomalsubunit101 shows that the toxin is unable to contactboth the bulged-G motif and the folded GNRA loopin the 50S ribosomal subunit at the same time owingto steric effects. Although it is not clear whether thetoxin selects an infrequently populated conformationor induces it, base flipping is required for toxinbinding and efficient cleavage. GNRA tetraloops arequite stable and the reported Tm for a C(GAAA)Gtetraloop is 65.9◦C, which corresponds to a �Gfoldof −4.2 kcal mol−1 at 37◦C.102 The thermodynamicstability is due to favorable H-bonding and stackinginteractions within the GNRA fold.11,55 Therefore,the unfolding of the stable loop structure in therestrictocin complex is very unusual.

The second example is the crystal structureof a ternary complex consisting of the histonemRNA hairpin, the unphosphorylated RNA-bindingand processing domain (RPD) of stem-loop-bindingprotein (SLBP), and the human exonuclease ERI135

(Figure 6). The mRNAs that encode all five corehistone proteins (H1, H2A, H2B, H3, and H4) areunique in that they are the only naturally occurringeukaryotic mRNAs that are not polyadenylated.103

Instead of a poly(A) tail, the histone pre-mRNA3′ untranslated region (3′ UTR) contains a 16-ntconserved stem-loop (Figure 6(d)) that is capped by aUYUM tetraloop and is the minimal substrate requiredfor high-affinity interaction with SLBP. The solutionNMR structure of the U(UUUC)A stem-loop from theUYUM family present in the histone H4-12 gene hasbeen solved by two laboratories in the absence of



(a) (b) (c)

(d) (e)

FIGURE 5 | Structure of restrictocin bound to the GAGA SRL tetraloop. (a) Summary schematic showing the hydrogen bonding and stackinginteractions observed in the free GAGA tetraloop (PDB code 430D). The sugar puckers and base configurations are color coded according to the keyshown in Figure 1(b). (b) Summary schematic showing the hydrogen bonding and stacking interactions observed in the GAGA tetraloop when boundto restrictocin (PDB code 1JBR). Protein side chains that make specific contacts with the tetraloop are depicted. (c) Superposition of the RNAbackbone and side chains of the free GAGA tetraloop (shown in cyan) and the unfolded GAGA tetraloop when bound to the toxin (shown in green).(d) Overall structure of restrictocin bound to the RNA hairpin. The protein is shown in purple cartoon and the RNA is shown in green. The position ofthe bulged-G motif relative to the tetraloop is highlighted. (e) Close-up of the protein–RNA-binding interface. The protein side chains are in gray andthe RNA ribose and bases are color coded as shown in (b).

SLBP9,10 (Figure 6(a)). These structures confirm thatthe six base pair RNA stem adopts a classic A-formhelix starting with the highly conserved G-C base pairat the base and extending up the stem to a loop-closingU-A base pair. The 5′ and 3′ flanking regions at thebase of the stem are disordered in solution. Thereare three unique features of the UUUC tetraloop.First, unlike the UNCG, GNRA, and CUUG classes oftetraloops, there is no evidence for a base pair betweenthe first and fourth nucleotides. The UUUC tetraloop isstabilized only by base-stacking interactions. Second,the conformation of the phosphate backbone in theloop is significantly different from that observed inmost tetraloops in that it lacks a sharp reverse turn andinstead reverses direction over multiple nucleotides.Third, a notable difference between the two NMRstructures is the orientation of U12 in the loop whichbase stacks against other loop uridines in one case9 butis solvent exposed toward the minor groove face in theother10 (Figure 6(a)). These observations likely reflectthe dynamic nature of the loop and its sensitivityto solution conditions because different salt and pHconditions were used in the two NMR studies.

Two proteins are known to bind this stem-loop:SLBP and an exonuclease, ERI1, that has been impli-cated in histone mRNA degradation.106 ERI1 is aDEDDh family exonuclease that trims 2–3 nt fromthe 3′ end of the mature histone mRNA stem-loop. Itbelongs to a family of structure-specific nucleases thatbind RNA hairpins or duplexes with a 2–3 nt 3′ over-hangs and has no sequence specificity for the RNA.107

The mode of interaction of SLBP withthe histone mRNA stem-loop has been wellcharacterized using biochemical and biophysical toolsby a number of laboratories. The contributionof specific RNA nucleotides to SLBP binding hasbeen quantified by mutagenesis using EMSA,10,108

nitrocellulose filter binding assays,104 and an eleganthigh-throughput microfluidics approach105 that takesinto consideration both structural and sequencefeatures of the RNA. These affinity measurementshave all been made using the Thr-171 phosphorylatedform of baculovirus-expressed SLBP (see below).The results indicate that the second G-C base pairand the sixth U-A base pair in the stem and twouridines in the loop (U12 and U14) are criticalfor interaction with SLBP. In addition, the flankingadenosines 5′ and 3′ to the stem-loop contribute to



(a) (b) (c)

(d)

(e) (f)

(h)

(g)

FIGURE 6 | Structure of the SLBP RNA-binding domain, ERI1, and histone mRNA hairpin ternary complex (PDB code 4HXH). (a) Summaryschematic showing the hydrogen bonding and stacking interactions observed in the solution nuclear magnetic resonance (NMR) structure of the freeUUUC tetraloops calculated by NMR. The structure of PDB code 1JWC9 is summarized on top and the structure of PDB code 1KKS10 is summarizedbelow. (b) Summary schematic showing the hydrogen bonding and stacking interactions observed in the UUUC tetraloop when bound to both SLBPand ERI1. SLBP protein side chains that make specific contacts with the tetraloop are depicted in orange, and ERI1 side chains that make specificcontacts with the tetraloop are depicted in black. (c) Superposition of the RNA backbone and side chains of the free UUUC tetraloop9 (shown in cyan)and the unfolded UUUC tetraloop when bound to both SLBP and ERI1 (shown in green). There is a loss of base stacking of all uridines in theprotein-bound complex and a reorientation of U13. (d) The sequence of the histone mRNA hairpin is shown. Nucleotides important for binding T171phosphorylated SLBP are highlighted in red and orange. G7 and U14 are critical for interaction with phosphorylated SLBP and are shown in red. (e)Overall structure of the SLBP RNA-binding domain (in orange), the histone mRNA stem-loop (in green), and ERI1 (in purple) ternary complex (PDBcode 4HXH). Helices in SLBP and the SAP domain are designated. (f) Close-up of the histone mRNA tetraloop (in green) bound to the SLBPRNA-binding domain (in orange) and ERI1 (in purple) in the ternary complex. Side chains in SLBP and the ERI1 SAP domain that contact the RNA aredesignated. (g) The structure of the ternary complex is color coded to show the variation in the Debye–Waller factor (or B-factor) in the protein andRNA components of the complex. Very high B-factors (shown in red) indicate regions of high mobility or where the electron density is more spreadout. Regions with low B-factors are shown in blue. Helix-B or SLBP has the highest B-factors in the ternary complex. (h) Crystal contacts observed inthe structure of the ternary complex are shown. The crystal shows extensive packing interactions between the nuclease domain of ERI1 from a binaryERI1/RNA complex (in yellow) that lacks SLBP, and the nuclease domain of a neighboring ERI1 molecule (in purple) from the ternary complex. TheSLBP RNA-binding domain is shown in orange. Helix-B of SLBP contacts the SAP domain of ERI1 from a neighboring binary complex. The RNA isshown in green (ternary complex) or cyan (binary complex).



SLBP binding (Figure 6(d)). In all studies, the UYUMtetraloop is a strong determinant of SLBP binding.Replacement of the UUUC tetraloop by an AUAC,GUUA, or a GNRA sequence results in a >200fold decrease in binding affinity toward SLBP.104

Replacement of the loop uridines U12 and U14 byany other nucleotide (including cytidine) stronglyreduces binding to SLBP.10,105 W-C base pairing ofthe closing UA base pair is also critical.10,104,105 Asthe functional groups of the tetraloop and closingbase pair are oriented toward the major groove,the generally accepted view is that the SLBP RPDwould recognize this face of the RNA hairpin, makingsequence-specific contacts with loop nucleotides.

NMR and mass spectrometry studies haveshown that human and Drosophila SLBP are intrin-sically disordered phosphoproteins.109,110 HumanSLBP is phosphorylated at 9 Ser/Thr residues in mam-malian cells111 and 23 Ser/Thr sites when isolatedfrom baculovirus.110 Solution NMR studies showthat the SLBP RPD is not stably folded in solution,has three helices, and undergoes a disorder-to-ordertransition upon RNA binding.112,113 The first 30amino acids of the SLBP RPD form a helix-turn-helixmotif in solution, but do not bind histone mRNAwith high affinity.112 A unique feature of SLBP isthat phosphorylation of a conserved threonine in a171TPNK sequence in the SLBP RPD is important forthe kinetics of interaction with histone mRNA.112,114

An apparent equilibrium dissociation constant (Kd)between 1.5 and 9 nM10,104,112,114 has been measuredfor the interaction of Thr-171 phosphorylated humanSLBP with the 26-nucleotide stem-loop. Baculovirus-expressed phosphorylated full-length SLBP has a slowassociation rate for the histone mRNA stem-loop(kon = 1.2 × 104 M−1 second−1) compared with thediffusion-limited rate of 1.0 × 109 M−1 second−1 anda very slow dissociation rate (koff = 5.7 × 10−4 min−1)yielding a binding constant (Kd) of 47.5 ± 17 nM asmeasured by surface plasmon resonance.112 The morebasic dephosphorylated full-length SLBP has a 10-foldfaster on rate toward the stem-loop, but a 100-foldfaster off rate of 7.44 × 10−2 M−1 second−1, yieldinga Kd of 528.6 ± 73 nM. This corresponds to anapproximately 11-fold decrease in binding affinity fordephosphorylated full-length dSLBP, which is similarto that reported using filter binding measurements.114

NMR studies provide evidence for cis-trans prolineisomerization about the 171Thr-172Pro sequence.112

Mutation of Pro-172 results in loss of RNA bindingand embryonic lethality.115 The 171Thr-172Prosequence is the binding site for the prolyl isomerasePin1 in vitro and in vivo.111

In the crystal structure of the unphosphory-lated SLBP RPD-histone mRNA hairpin-ERI1 ternarycomplex,35 the SLBP RPD (residues 125–223) con-sists of three helices: helix-A (Glu-132-Lys-146),helix-B (Ile-149-Val-158), and helix-C (Arg-180-Phe-196) (Figure 6(e)). Consistent with previousreports,110,112,113 a significant portion (57 of 99residues in total) of the RPD is disordered, with 37residues having no visible electron density and 20residues in loops that are likely flexible given their highB-factor. The site of phosphorylation, Thr-171, is ina flexible loop between helix-B and helix-C and is ori-ented away from the RNA (Figure 6(e)). Helix-C is therecognition helix and it approaches the major grooveface of the RNA hairpin from the 5′ end, but does notlie in the major groove. Helix-C acts as a molecularruler and extends from the base of the RNA stem tothe top of the tetraloop. A specific base contact is madebetween the side-chain guanidinium group of Arg-181and the second guanine base (G7) of the RNA stem,consistent with previous biochemical studies.104,105

Surprisingly, the interaction of SLBP with thetetraloop consists of nonsequence specific π -stackinginteractions between Tyr-144 of helix-A and the firsturidine (U12) of the loop and His-195 with the non-conserved cytidine, C15 (Figure 6(b) and (f)). Substitu-tion of C15 with any other nucleotide has no apprecia-ble effect on SLBP binding.10,105 Tyr-144 also showsa T-shaped stacking interaction with U14 in the crys-tal structure.35 The RNA tetraloop is unfolded in thecrystal structure when compared with the two solu-tion NMR structures of the free RNA hairpin (Figure6(a)–(c)). The first, third, and fourth bases of thetetraloop are flipped out and oriented toward SLBP,whereas the second uridine (U13) is oriented towardthe SAP domain of ERI1 and interacts with Tyr-66,Phe-61, and Lys-111 of ERI1 (Figure 6(f)). The ERI1nuclease approaches the RNA hairpin from the 3′ endwith the SAP domain interacting with the tetraloopand nonspecific contacts are made with the backboneof the stem and the 3′ flanking region (Figure 6(e)).

Although the structure is the first exampleof a ternary complex involving two proteins incontact with an RNA hairpin, there are severalinconsistencies with biochemical studies to beresolved. For example, the lack of sequence-specificcontacts between SLBP and the tetraloop in the crystalstructure is not consistent with biochemical studiesof the phosphorylated SLBP–histone mRNA binarycomplex. It is possible that the orientation of helix-Bis different in the context of the phosphorylatedSLBP–histone mRNA complex. The solution NMRstructure of residues Glu-129-Val-158 indicates thepresence of two helices encompassing Glu-132-Ile-142



and Thr-147-Val-158. These helices are longer [helix-A (Glu-132-Lys-146) and helix-B (Ile-149-Val-158)]in the RNA-bound crystal structure. In the crystal,helix-B is almost perpendicular to helix-A, and liesfarther away from the RNA-binding interface in thecrystal. Helix-B has the highest temperature factors(B-factors) in the entire ternary complex (Figure 6(g)),which could be indicative of high-amplitude motionsfor the peptide backbone atoms of this helix. Helix-B isalso involved in crystal contacts with the SAP domainof a neighboring ERI1 molecule in the lattice (Figure6(h)). Extensive crystal contacts are also observedbetween the nuclease domains of ERI1 (Figure 6(h)).The effect of these contacts on the conformation ofthe tetraloop is unclear. Future structural studies bycrystallography and NMR on the binary and ternarycomplexes should help clarify the mode of recognitionof the histone mRNA hairpin in detail.

CONCLUDING REMARKS

We have described the different modes of RNAtetraloop recognition by proteins as observed in thefew complexes that have been structurally character-ized in detail. The mechanisms include specific baserecognition by a hydrophobic pocket, adaptive bind-ing of a helix in the major groove of a GNRAfold, shape-specific recognition of the (A/U)GNNtetraloop minor groove by a helix, simultaneous fold-ing and binding of an unfolded tetraloop, and recog-nition by tetraloop unfolding. There are examplesof disordered-to-ordered transitions where the pep-tide is conformationally dynamic and adapts itsstructure to that of the RNA target, and ordered-to-

disordered transitions that involve the RNA tetraloop.In most cases, the structural characteristics of the RNAtetraloop motifs are conserved in the protein–RNAcomplexes. The five modes of recognition we havedescribed do not exhaust the possibilities, and, nodoubt, further studies will reveal additional mecha-nisms of recognition.

To fully comprehend the modes of RNAtetraloop recognition by proteins we need to under-stand the contribution of dynamics to recognition.Studies using NMR, time-resolved fluorescence, andmolecular dynamics simulations have shown thattetraloops, although stable, populate several minoror unfolded states at equilibrium. How dynamics ofthe RNA and protein contributes to complex forma-tion has been relatively unexplored. Another area thathas not been well studied is the mode of RNA recog-nition in larger ribonucleoprotein complexes wheremore than one protein can recognize the tetraloopsimultaneously. The SLBP-ERI1-histone mRNA stem-loop is the only example of a ternary complex forwhich one structure is available, and in that case, theRNA tetraloop adapts to accommodate both proteinsin the complex. Therefore, additional structural stud-ies are required on larger assemblies. Finally, a numberof proteins are posttranslationally modified by phos-phorylation, ubiquitination, acetylation, etc. SeveralRNAs, especially rRNAs, are also edited or undergobase modifications. Such modifications are importantfor proper function, but structural information onmodified proteins or RNAs in complexes is currentlylacking. We anticipate that these are the areas thatwill lead to new insights into the diverse modes ofRNA tetraloop–protein interactions in the future.

ACKNOWLEDGMENTS

Supported by NIH 1RO1-GM076660 and 1RO1-GM076660-04S1 (ARRA) grants (R.T.) faculty start-upfunds from HWI to R.T. and by NIH 1RO1-GM73969 (E.P.N.). Support for A.P.D. was provided by NationalInstitutes of Health Training Grant 2T32 GM008280. We gratefully thank Dr Pascale Legault (University ofMontreal, Canada) for PDB coordinates of the λN-peptide-λ boxB RNA complex. We also thank the reviewersfor their insightful comments. Our goal was to focus on the most well-studied examples and structures thatdepict a unique mode of molecular recognition. We apologize to individuals whose work we could not includeowing to space limitations.

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Recognition modes of RNA tetraloops and tetraloop-like motifs by RNA-binding proteins