MINIREVIEW

Anna Marie Pyle

Metal ions in the structure and function of RNA

Received: 29 March 2002 /Accepted: 4 June 2002 / Published online: 18 July 2002� SBIC 2002

Abstract It is becoming increasingly clear that RNA ismore than a passive carrier of genetic information. FoldedRNA molecules play key roles in almost every aspect ofcellular metabolism, including protein transport, RNAsplicing, peptide bond formation, and translational reg-ulation. This is facilitated by themultifunctional nature ofRNA biopolymers which can serve as rigid structuralscaffolds, conformational switches, and catalysts forchemical reactions. In all cases, metal ions play a crucialrole in RNA function. For folded RNA molecules, thepathway for adopting proper tertiary structure, and thestabilization of that structure, depends on specific andnonspecific interactions with certain classes of metal ions.There is a rapidly expanding repertoire of RNA structuralmotifs that typically sequester metal ions, and these arebeing studied using new spectroscopic and chemicalmethodologies. Many ribozymes (catalytic RNA mole-cules) depend onmetal ions as cofactors that are explicitlyinvolved in the chemical mechanism of catalysis. All ofthese functions are exemplified by recent studies of groupII introns, which are among the largest ribozymes foundin Nature. In this case, there are specific roles for metalions in the folding pathway, the tertiary structure and thechemical mechanism.

Keywords Ribozyme Æ RNA Æ Folding Æ Catalysis ÆMagnesium

Introduction

Recent investigations have revealed that RNA is in-volved in a tremendous diversity of biological processes.It functions not only as a passive informational molecule

(in the form of mRNA), but also as a precisely foldedpolymer that can act as structural scaffold, regulatorysignal, or catalytic enzyme. Major examples of func-tional RNA molecules include ribosomal RNA (rRNA),which catalyzes and regulates protein synthesis, smallnuclear RNAs (snRNAs), which are involved in splicingof introns in the nucleus, the signal recognition particle(SRP), which transports proteins through membranes,and the many ribozymes that catalyze reactions on bothRNA and DNA (such as group I introns) [1, 2, 3]. In allof these cases, metals play an essential role in the ar-chitectural assemblies and catalytic mechanisms that arecentral to RNA biology. Perhaps more than any othertype of biomolecule, the behavior of RNA is inextricablylinked to the function of metal ions and to the basicprinciples of bioinorganic chemistry (Fig. 1).

RNA folding pathways and the influence of metals

Folded RNA molecules adopt highly complex structuresthat are globular, like the form of many functionalproteins (Fig. 2). A typical misconception is that RNA isa stringy, single-stranded molecule that does not adopt adefined architecture. In fact, most functional RNAmolecules are compact, stable, and tightly folded into aunique conformation [4, 5, 6, 7]. It is notable that evenmRNA molecules (which are probably required to besomewhat single-stranded in order to be read by the ri-bosome) contain structured terminal regions that serveas signals for regulating gene expression [8, 9, 10]. Giventhe complexity of RNA structure, what are the foldingpathways of RNA and how are metal ions involved inthe process?

RNA folding is typically believed to approximate atwo-stage process. The first stage is the formation ofRNA secondary structure (resulting in regions of single-and double-stranded character). The adoption of sec-ondary structure can be stimulated by almost anythingthat screens charge and compensates for the electrostaticpenalty of bringing polyanionic backbones into

J Biol Inorg Chem (2002) 7: 679–690DOI 10.1007/s00775-002-0387-6

A.M. PyleDepartment of Biochemistry and Molecular Biophysics andThe Howard Hughes Medical Institute; Columbia University,630 W. 168th Street, New York, NY 10032, USAE-mail: amp11@columbia.eduTel.: +1-212-3055430Fax: +1-212-3051257

proximity [11, 12]. It is therefore stimulated by thepresence of monovalent cations, divalent cations, cat-ionic polyamines, and even basic proteins (Fig. 3).

The second stage is the adoption of RNA tertiarystructure, in which secondary structure collapses into athree-dimensional network of stacked duplexes and un-usual intramolecular interactions comprising both baseand backbone residues [13]. Unlike secondary structure,the formation of RNA tertiary structure is governed bystringent metal ion requirements [14]. Divalent cations

such as magnesium are usually strictly required; in a fewcases, potassium makes important contributions [15].

Many RNA molecules have a very rugged foldinglandscape, in which stable off-pathway intermediatescompete with formation of the native state. It is often therate constant for unfolding of off-pathway intermediatesthat dictates overall rate constants for folding (a kinetictrap) [16]. By contrast, there are now several examples ofRNA molecules {namely a subsection of ribonuclease P(RNAse P) [17] and a ribozyme derived from a group IIself-splicing intron [18]} that fold directly to the nativestate. In these cases, the concentration of Mg2+ and thestage at which it is introduced into the folding reaction arecritical for a smooth folding pathway (Fig. 3). In general,the formation of RNA tertiary structure is dependent onfour parameters: (1) RNA sequence; (2) metal ion identity(typically Mg2+ versus Ca2+ and K+ versus Na+); (3)metal ion concentration; (4) the presence of RNAbindingproteins and polyamines.

Metal ions and the stabilization of RNA tertiary structures

Types of metal ions in RNA

While a variety of different metals contribute to RNAfunction, certain ones are strongly preferred [13, 14, 19,20]. Among monovalent ions, K+ is by far the mostcommon and it is often specifically required [15, 21].Na+ and Li+ can contribute to the folding of certainRNAs [22], although they can also poison other systemsby competing for potassium [23]. Mg2+ is the mostcommon ion involved in RNA folding [14, 24] and it isoften required for catalysis as well [25]. In limited cases,

Fig. 1. The high-resolution crystal structure of ribosomal S15protein bound to its rRNA site. The purple spheres are Mg2+. Theblue and yellow spheres are proposed to be Na+ and K+,respectively. From PDB file 1clk1

Fig. 2. Comparing globular protein and RNA enzymes of similarsize. Like cyclooxygenase (pdb 1prh; 63,783 Da), this dimericstructure of a group I intron core (pdb 1grz; 168,782 Da for dimer)folds into a distinctive globular structure with specific interior andexterior regions

Fig. 3. A simplified view of the complex folding pathway for RNAmolecules, and the involvement of metal ions. The formation ofsecondary structure is governed by counterion condensation andcan therefore be promoted by monovalent ions (particularly K+).The formation of tertiary structure and intermediates usuallyrequires Mg2+. A single off-pathway intermediate in equilibriumwith the other states is shown as an example. Note that obligate on-pathway intermediates can also be populated during folding to thenative state (not shown). Adapted from [93, 94]

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Ca2+, Mn2+, and even Cd2+ can substitute for Mg2+,although these are rarely the natural ionic cofactors [26,27]. Trivalent ions (other than organic polyamines,which are abundant in cells) are not natural cofactors ofRNA, although they are useful structural probes. Tb3+

and other lanthanides are good probes of Mg2+ bindingsites [28], which are revealed through RNA cleavage(hydrated Tb3+ acts as a strong general base) [29], or byluminescence [30]. Cobalt(III) hexammine has also beena valuable probe for evaluating metal ion bindingthrough outer-sphere mechanisms [24, 31].

Types of metal ion binding in RNA

The different types of metal ion binding in RNA aredepicted in Fig. 4 [24, 32].

Diffuse binding

Described classically by Manning and Record [11, 12],and more recently using a Poisson-Boltzmann treatmentof electrostatics [33, 34], this mode of binding providescharge screening that overcomes electrostatic repulsionbetween RNA backbone segments (Fig. 3, secondarystructure). It can be provided by a diversity of cationsand is essential for both secondary and tertiary structureformation.

Site-bound, outer sphere

Mg2+ often binds to specific sites without forming directcontacts between RNA functionalities and the metal ion(Fig. 4A) [24, 35, 36]. Instead, the water ligands of mag-nesium hexahydrate interact with RNA base and back-bone substituents to stabilize specific RNA motifs [37].

Despite expectations for a rapid water exchange rate,these outer-sphere contacts are thermodynamically sig-nificant [37]. The major groove edge of guanosine(through the O6 and N7 atoms) is a particularly commonligand for magnesium-bound water. In cases where thistype of binding occurs, theMg2+can typically be replacedby cobalt(III) or osmium(III) hexammine, which are ex-change-inert mimics of hydrated Mg2+ [24, 35]. In con-trast to this specific type of metal complex recognition,there are cases of strong site binding that occur withoutthe formation of any contacts to the metal or to its inner-sphere ligands. In these cases, the metal ion interacts atsites of highly localized negative electrostatic potential[34, 38]. As such, the magnesium can often be replaced byorganic polyamines such as spermidine.

Site-bound, inner sphere

Direct contacts between RNA and Mg2+ are often im-portant for RNA structure and catalytic function(Fig. 4B) [33, 39]. The most common ligands on RNAare the phosphoryl oxygens, the purine N7 heteroatoms,base keto groups (particularly O6 of guanosine and O4of uracil), and the ribose 2¢-OH. Crystal structures haverevealed that these direct contacts commonly displace 1–3 water ligands on the hexacoordinate Mg2+ [39, 40].Biochemical experiments using nucleotide analog inter-ference mapping (NAIM) have shown that these directcontacts are thermodynamically significant and they canusually be probed by metal ion specificity switches [21,41]. For example, direct contact between a phosphoryloxygen and Mg2+ can often be replaced by a functionalinteraction between a phosphoryl sulfur and thiophilicCd2+ [42, 43]. Similarly, direct contacts between RNAoxygen ligands and potassium have been revealed bothcrystallographically and biochemically by substitutingoxygen with sulfur and K+ with thallium(I) [21].

Regions of metal ion binding in RNA

In RNA duplexes (and also in A-form DNA duplexes),metals bind in the deep, narrow major groove.

Fig. 4A, B. Some types of site binding in RNA tertiary structure.A Interaction between hydrated Mg2+ and nucleobases throughouter-sphere (water-mediated) interactions. B Types of inner-sphere (direct) interaction between Mg2+ and phosphoryl groupsof the backbone (left) and base functionalities (right). Adaptedfrom [95]

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Calculations of electrostatic surface potential reveal thatthe major groove has a pronounced negative potential(Fig. 5). Both crystal structures and NMR studies haverepeatedly shown that the region is a deep sink for metalion binding [44].

There are many types of specific metal binding motifsin RNA tertiary structure. One of the most common isthe major groove of tandem G-U pairs (Fig. 6). Thismotif creates a cavity that is lined by lone pairs, whichcreate an unusually negative potential and provide goodligands for metal ions and coordinated water molecules[36]. Internal loop structures in RNA often bind metalions, the most striking example being the loop E motif,which actually binds Mg2+ as a binuclear cluster thatsqueezes neighboring backbones into unusually closeproximity (Fig. 7) [40]. One of the most intriguing metalion binding sites is the A-platform motif, which specifi-cally binds a dehydrated potassium ion. This network ofstrong, direct contacts between RNA and potassiumhave been observed crystallographically and biochemi-cally, by switching a critical guanosine residue with 6-thioguanosine, which disrupts function until a fractionof K+ is replaced with thallium(I) (Fig. 8) [21].

The role of metal ions in RNA catalysis

There are three different families of natural ribozymesthat are known at the present time: (1) the large phos-phoryl transfer ribozymes; (2) the small phosphoryltransfer ribozymes; (3) aminoacylesterase ribozymes.

Each group has chemically distinct mechanistic featuresthat result in different types of metal ion requirements.

The large phosphoryl transfer ribozymes

This group of enzymes includes the self-splicing group Iand group II introns, ribonuclease P, and, perhaps, theeukaryotic spliceosome. They cleave or ligate the phos-phodiester linkages of RNA (and often DNA) through aclassical SN2 mechanism involving in-line attack of anexogenous nucleophile that is typically an alcohol orwatermoiety. Thismechanism requires relatively complexactive sites for orienting and activating both nucleophileand leaving groups, as well as activation of the scissilephosphate. To meet these electrostatically and stericallydemanding tasks, this class of ribozymes appears todepend strongly on divalent metal ions. More than anyother group of ribozymes, the large phosphataseribozymes are considered obligate metalloenzymes [25].

The Tetrahymena ribozyme, which is derived from agroup I intron, requires magnesium ions to stabilize itstertiary structure [5, 45] (see Fig. 2, right, for foldedstructure) and to promote catalysis [46, 47, 48]. Studieson the metal ion requirements of this ribozyme resultedin the first applications of the ‘‘metal ion rescue experi-ment’’ [41, 47], which showed that specific ribose andphosphoryl oxygen atoms were important ligands fordirect divalent metal ion coordination in the ribozymeand, specifically, in the transition state for chemicalreaction.

The metal ion rescue experiment is far from foolproof[49, 50, 51], particularly in cases where rescue cannot beachieved by more highly thiophilic ions such as Cd2+

and Zn2+, and where sulfur substitution (even after

Fig. 5. The electrostatic surface potential of RNA duplexes, whichhave the A-form helical configuration. Red regions have a largenegative potential (maximum –15 kt/E on this scale), while whiteregions are neutral. Blue regions have a positive potential(maximum +15 kt/E on this scale). Adapted from [96]

Fig. 6. The tandem G-U pair motif for binding divalent metal ions(upper right). The motif involves interactions between O6 and N7 ofguanosine, which are a common site for direct and outer-spheremetal ion coordination (lower right). The motif has been studiedcrystallographically [36], one strand of which is shown interactingwith Os(III)(NH3)6 (left), adapted from [36]

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coordination) leads to significant alteration of local hy-dration and electrostatics [52]. Nonetheless, there arenumerous cases in which the experiment has providedconvincing evidence for specific metal ion coordination[97]. RNA metalloenzymes typically depend on spec-troscopically silent metal ions. Therefore, chemical mu-tagenesis experiments such as the metal ion rescueexperiment represent one of the most informative toolsfor investigating their structure and mechanisms.

In one of the first examples of this experiment, anoligonucleotide substrate for the Tetrahymena ribozymewas synthesized in which the 3¢-hydroxyl leaving groupat the scissile linkage was replaced with a 3¢-thioylgroup. While this prevented activity in the typicalMg2+ cofactor, the substrate was reactive and cleaved

in the presence of Mn2+, Cd2+, or Zn2+ (Fig. 9) [46,47]. Rescue with Zn2+ was particularly pronouncedwhen sulfur was introduced as a double substitution atboth the 3¢- and pro(Sp) phosphoryl positions of thescissile linkage (defined in legend, Fig. 9) [43]. Theseexperiments suggest that a metal ion interacts directlywith the leaving group and with the pro(Sp) oxygen inthe trigonal bipyramidal transition state, helping toreduce the dramatic build-up of negative charge, andpotentially providing architectural support for thetransition state.

By combining similar rescue experiments with newmethods for monitoring the binding constant of specificmetal ions, it has been possible to create a detailed,functional picture of metal ion participation in thechemical step of group I intron ribozymes. Taken to-gether, these studies suggest that at least three distinctmetal ions participate in the reaction, which includescoordination to both the 2¢-and 3¢-groups of the nucle-ophile (Fig. 10) [48, 53]. This is one of the most detailedsnapshots of metal ion involvement in catalysis by anyenzyme, and thereby demonstrates that ribozymes canprovide fundamentally novel insights into mechanismsof biomolecular catalysis.

Metal ions play an essential role in chemical catalysisby RNase P [54, 55], group II intron ribozymes (vidainfra) [42, 56], and even the spliceosome [57, 58, 59],which have been studied using techniques that are sim-ilar to those employed for the Tetrahymena ribozyme. Inthe latter three cases, direct metal coordination to theleaving group appears to be particularly important.Metal ion coordination to scissile phosphoryl oxygensand to the respective nucleophiles differs among thesystems, suggesting that there are multiple ways forRNA to promote catalysis even within the same mech-anistic family.

Fig. 7. The loop E motif bindsmagnesium in a variety offorms. Crystallographic investi-gations of the loop E motif(lower right) have revealed anexceptionally narrow majorgroove that is bridged by un-usual arrangements of magne-sium ions (left), two of whichform a binuclear cluster (upperright) [40], adapted from [40]

Fig. 8. The A-platform motif binds a dehydrated potassium ion.In this motif (which is not restricted to adenosines), a pair ofnucleotides have slipped such that the downstream base lies notbeneath the plane of its partner but next to it, in a side-by-sidearrangement (see green bases, at left). Together with conserved,surrounding nucleotides (such as the G, in red), this motifcoordinates potassium ions through numerous inner-sphere con-tacts. Adapted from [21]

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The small phosphoryl transfer ribozymes

This group includes the hammerhead family, the hairpinribozyme, the hepatitis delta ribozymes, and the Varkudsatellite (VS) ribozyme (Fig. 11) [60, 61, 62]. These areself-cleaving RNA motifs that are typically found in thegenomes of primitive viroids and viruses, where they cutlong genomic RNA strands into individual genes [63].They share a distinctive mechanism involving an en-dogenous nucleophile that is located within the scissile

linkage itself. In these ribozymes, an activated 2¢-hydroxyl group at the cleavage site attacks the adjacentphosphate moiety, resulting in cleavage of the strand togenerate 2¢-3¢ cyclic phosphate and 5¢-hydroxyl termini.Because these ribozymes require a ribose at the cleavagesite (unlike the large phosphoryl transfer family dis-cussed previously), they can only cleave RNA molecules.

Although these ribozymes appear to share a commonmechanism, there are profound differences in the waythat they catalyze strand scission. The role of divalentmetal ions in the mechanisms of these ribozymes is oneof the major ways that they differ. Divalent metal ionshave been implicated in chemical mechanism of thehepatitis delta virus ribozymes [64, 65], the hairpin ri-bozyme [66, 67], and the hammerhead ribozyme [68, 69].While all of these ribozymes tend to function optimally(and are generally studied or applied) in the presence ofMg2+, recent work has established that the hairpin,hammerhead, and VS ribozymes retain activity underconditions where divalent cations are replaced by molarquantities of monovalent cation, exchange-inert metals,or even organic polyamines [65, 68, 70, 71, 72]. Thesefindings establish that divalent metal ions are not strictlyrequired for RNA catalysis by this family of ribozymesand that functionalities on the RNA (such as nucleo-bases with shifted pKa values) play an importantrole in transition-state stabilization (Fig. 12) [64, 73].

The absence of a strict Mg2+ requirement for thisfamily of ribozymes is often narrowly interpreted asevidence that metals have no involvement in the catalyticmechanism of these ribozymes [65, 70, 71]. This isprobably an overstatement, given the tremendous elec-trostatic stabilization that is required for nucleophilicreactions at phosphorus. Divalent metal ions can stabi-lize packed RNA tertiary structures without undergoingany inner- or outer-sphere coordination [34], and it ispossible that they can similarly provide electrostaticstabilization of a transition state without forming anetwork of elaborate contacts (see diffuse ‘‘n+’’ inFig. 12) [67, 68, 72]. When metal ions interact with anRNA site or stabilize a transition state in this manner, itshould not be surprising that they can be replaced invitro by molar quantities of monovalent ion [67], co-balt(III) hexammine, or an organic polyamine, as hasbeen observed in studies of RNA folding [33, 34, 38].

Fig. 9. A metal ion rescue experiment for probing inner-spherecoordination between a magnesium ion and the 3¢-hydroxyl groupof a phosphodiester linkage. When the 3¢-moiety is replaced bysulfur, Mg2+ no longer coordinates and function is lost. Functionis regained in the presence of thiophilic metal ions that can interactwith sulfur. Note that the phosphoryl center can be consideredprochiral, with the individual oxygen atoms denoted pro(Rp) andpro(Sp) in configuration (not indicated in this figure). This isparticularly relevant when one of the oxygens is replaced by sulfur,or in the context of a helix, in which the chemical environment ofthe two atoms differs

Fig. 10. The proposed transition state for phosphodiester cleavageby the Tetrahymena ribozyme. At least three metal ions (labeledMA–MC) are proposed to interact at the sites shown duringcatalysis

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The aminoacylesterases

The notion that RNA could catalyze the making andbreaking of amide bonds was supported by the in vitroselection of new ribozymes with completely novel cata-lytic capabilities that included a variety of reactions at

carbon. The ability to react at carbon was confirmedwhen a Tetrahymena ribozyme-substrate complex wasspecifically engineered to place an activated aminoacylester at the scissile bond [74]. This novel substrate wasreadily cleaved in the presence of Mg2+, suggesting asimilar mechanism for both phosphoryl and carbonyl

Fig. 11. Secondary structuresof three small phosphodiester-ase ribozymes. Arrows indicatesites of cleavage. The bottompanel shows how the self-cleav-ing hammerhead motif has beenre-engineered into a ribozymefor targeting and cleavingexogenous RNA substrates

Fig. 12. Schematic of somecatalytic mechanisms for smallphosphodiesterase ribozymes.The general base for deproto-nating the nucleophilic 2¢-OHgroup could be an adenosineN1 (or other nucleobase) with ashifted pKa, causing the base tobehave like a histidine side-chain. Similarly, pKa-shiftedcytosine has been invoked as ageneral acid for protonating theleaving group. Still unclear (andstill a potential role for metals)is the mechanism for electro-static stabilization in the tran-sition state (shown as a redcircle adjacent to the n+)

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activation. However, none of these examples representsthe normal chemical behavior of a natural ribozyme.

This changed when biochemical [75], mutational, andstructural studies of the ribosome converged to dem-onstrate that the synthesis of proteins is actually cata-lyzed by the RNA of the ribosome [76]. The peptidyltransfer site is composed exclusively of RNA; ribosomalproteins lie on the exterior of the 50S subunit, awayfrom the active site and from the interface with the 30Ssubunit. Although there is a requirement for Mg2+ inribosomal function, it is currently not clear if it isstructural, regulatory, or catalytic. The involvement ofmetal ions in catalysis by the ribosome has thereforebecome a fascinating issue, and one which can now beexplored through a variety of biophysical approaches.

A case study: the role of metal ions in group II introns

One of the most important roles for catalytic metal ionsin biology is in the chemistry of RNA splicing. Allbiomolecular machines that catalyze RNA splicing aredependent on Mg2+ for both folding [5, 77] and chem-ical catalysis [47, 56, 57, 59]. RNA splicing is defined inthe following way. Most eukaryotic genes are tran-scribed into mRNA molecules that are not ready fortranslation into protein. Before translation, mRNAmolecules (and most tRNA, rRNA, snRNA, andsnoRNA molecules as well) undergo the process ofsplicing, in which noncoding introns are removed fromcoding exons [78, 79]. It is staggering to consider thecombinatorial implications of pre-mRNA splicing in

humans, in which an average mRNA contains �10 ex-ons that may or may not be spliced, depending on thetissue or developmental stage of the individual.

There are four different machines that catalyze RNAsplicing [80]: (1) group I introns, which are autocatalyticintrons that splice themselves out of precursor RNAs;(2) group II autocatalytic introns; (3) the eukaryoticspliceosome; (4) the tRNA splicing (protein catalyzed).The first two examples are ribozymes, and it is nowsuspected that RNA components of the eukaryoticspliceosome (which processes almost all human genes)are catalytic components of that machine [81, 82].Therefore, for the most part, the splicing of RNA iscatalyzed by RNA.

The author’s research group has focused on thestructure and chemical mechanism of group II self-splicing introns. These large ribozymes are organizedinto six domains of secondary structure, which fold intoa tertiary structure that recognizes 5¢-exon (or oligonu-cleotide substrates) through two stretches of base pairing(see green and blue lines, Fig. 13) [83, 84]. The nucleo-phile in the first step of splicing is the 2¢-OH group of abulged adenosine in domain 6 (D6), which reactsthrough the mechanism described previously for largephosphoryl transfer ribozymes (Fig. 13). Group IIintrons are true metalloenzymes that require Mg2+

specifically for folding [77, 85], substrate binding [86],and for direct coordination to the leaving group duringchemical catalysis [42, 56, 57]. Despite their great size(usually >1000 nucleotides), group II introns can bestudied because their constituent domains can besynthesized separately and then reacted together in

Fig. 13. Schematic of the sec-ondary structure and self-splic-ing pathway for a group IIintron. Group II introns recog-nize their 5¢-exons (or oligonu-cleotide substrates) throughbase pairing interactions be-tween the IBS1-EBS1 and IBS2-EBS2 regions (blue and greenlines, respectively, top panel).The pathway for self-splicinginvolves a lariat RNA (bottompanel)

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reconstitution experiments that evaluate the role of in-dividual motifs and even individual atoms in the struc-ture [84]. These studies have shown that domain 5 (D5)forms the active-site center of the enzyme (Fig. 14) [77,87], and that individual atoms on D5 have specificmechanistic roles (Fig. 15) [88, 89, 90]. A variety ofbiochemical and spectroscopic experiments have shown

that a metal ion binding platform within D5 plays aninstrumental role in catalysis.

Tb3+ as a probe of Mg2+ bindingin a group II intron ribozyme

In order to obtain a snapshot of metal ion binding sitesthroughout the group II intron, we employed Tb3+ as aprobe. Tb3+ is an excellent mimic for Mg2+ because thetwo ions share similar ionic radii and coordinationpreferences [28, 85]. In addition to being a luminescention, Tb3+ has the added advantage of promoting strandscission at sites of strong binding in RNA. This may bedue to the fact that hydrated Tb3+ has a pKa of 7.9,making it a potential general base for deprotonatingadjacent 2¢-hydroxyl groups and thereby facilitatingspecific RNA cleavage.

Working with a folded group II intron ribozyme thatwas 32P-labeled at either the 5¢- or the 3¢-terminus, Tb3+cleavage revealed many of the major sites for strongmetal ion binding within the intron [85]. Most of thesesites correspond to regions that surround the substratecleavage site and/or are already implicated in the cata-lytic mechanism. In particular, Tb3+ cleavage of D5provided the first evidence for strong metal ion bindingwithin the D5 internal loop (or elbow region) that haspreviously been implicated in reaction chemistry(Fig. 16). Remarkably, direct metal ion binding has beenobserved at the same location within an analogousstructure that promotes activity of the eukaryoticspliceosome [58], thereby providing convincing evidencethat group II introns and the spliceosome share mech-anistic features and, potentially, a common ancestry.Consistent with a recent crystal structure of D5 (andwith ion binding of isolated tetraloops in general) [91],

Fig. 14. Tertiary architecture of a group II intron core. Usingfunctional distance constraints from chemogenetic experiments, amodel of the core was constructed in which D5 (red) is surroundedby a tripod of interactions involved in ground-state binding (green),chemistry (yellow), and fidelity (blue) [77]

Fig. 15. The two faces of groupII intron D5. The binding face(left) contains atoms that par-ticipate in ground-state bindingto receptor motifs in D1 (j-j¢and f-f¢, as shown). The chem-ical face (right) on the oppositeside of D5 contains atoms thatparticipate in reaction chemis-try and metal ion binding

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Tb3+ cleavage at the D5 tetraloop is also observed.However, unlike strong Mg2+ binding and Tb3+

cleavage in the elbow motif, Tb3+ cleavage of the tet-raloop is significantly reduced when D5 is bound to therest of the intron [85].

NMR studies of Mg2+ binding to D5

Owing to its mechanism of RNA cleavage, Tb3+ canonly reveal a subset of important ion binding motifs. Itis unable to cleave phosphodiester linkages that liewithin an RNA duplex (such as the major groove ionbinding motifs discussed previously). Duplex regionsconstrain the phosphodiester linkages so that 2¢-hy-droxyl groups cannot assume the proper conformationfor in-line attack on adjacent phosphate groups.

To visualize all of the metal ion binding sites withinD5, and to obtain a high-resolution solution structure ofthe domain, we have studied D5 by NMR. Two types ofexperiments have been particularly helpful in revealingdivalent ion binding sites within the isolated domain.Mg2+ ion binding results in specific proton chemicalshift changes at neighboring residues (Fig. 17). Similarly,paramagnetic line broadening by Mn2+ reveals sites ofdivalent metal ion interaction. Like Tb3+ cleavage andcrystallographic studies, Mg2+ and Mn2+ titrationsimplicate metal ion binding at the tetraloop and adjacentto the elbow bulge. Unlike the other methods, NMRexperiments also reveal strong divalent ion binding inthe major groove of stem 1, which contains the ‘‘cata-lytic triad’’ of conserved AGC nucleotides (Fig. 18). Inthree-dimensional space, major groove nucleotides ofthe elbow loop and the AGC triad are in close proximity(Fig. 15, right), which is intriguing given that both re-gions contain the most important atoms in group IIintron chemistry. Taken together, these data suggestthat the AGC triad and the elbow loop form a metal ionbinding platform that participates directly in chemicalcatalysis. Remarkably, this function of D5 was predicted

Fig. 16a, b. Tb3+ cleavage of group II intron D5. a Polyacryla-mide gel showing sites of Tb3+ cleavage within D5, all of which arecompeted away by Mg2+ (not shown). b A schematic showing thelocations of cleavage on a D5 secondary structure. The strongestsite (red) remains intense even when D5 is bound within the intron,while the weaker tetraloop site (orange) disappears in the bound,active state

Fig. 17. Changes in D5 proton chemical shift patterns duringMg2+ titration experiments. Strongest positions of chemical shiftare numbered with respect to position in D5 [considering that G815(see Fig. 16) is G1 of D5]

Fig. 18. Summary of metal ion binding sites in D5. Yellow arrowsindicate binding sites that were revealed by NMR experiments; bluearrow indicates site observed during Tb3+ and crystallographicexperiments; white arrow indicates a site that is occupied only infree D5 (not in the folded intron). Red letters indicate nucleotidesthat contain atoms with effects on the chemical step of reactions

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long ago, by analogy to studies on protein enzymes andthe eukaryotic spliceosome [92].

Conclusions

Studies with group II introns and other ribozymes haverevealed that Mg2+ and K+ can play an essential role infolding, molecular recognition, and catalysis by RNA.Although RNA function is more dependent on metalions than any other type of biopolymer, it has receivedrelatively little attention by the community of research-ers in bioinorganic chemistry. In large part, this is be-cause RNA interacts with metals that arespectroscopically silent and do not lend themselves toevaluation by techniques that have evolved for studyingprotein metalloenzymes. Studies of metal ion-RNA in-teraction are further challenged by the fact that, in ad-dition to strong, specific metal ion sites, each RNA alsocoordinates a multitude of wealdy-bound metal ions forcharge screening. New approaches for studying the bi-oinorganic chemistry of RNA are being developed andthese include adaptations of NMR, ESR, lanthanideprobing, chemogenetic metal ion rescue experiments,and crystallography. However, there remains a need forinnovation and the application of novel approaches formonitoring the function of Mg2+ in both protein andRNA enzymes. Only when we can visualize and trackMg2+, Ca2+, and K+, which are by far the most com-mon metal ions in biological systems, will we develop abalanced understanding of metal ions as they function innature.

Acknowledgements This work was supported by the National In-stitutes of Health and the Howard Hughes Medical Institute.

References

1. Gesteland RF, Atkins JF (1993) The RNA world. Cold SpringHarbor Laboratory Press, Cold Spring Harbor, NY

2. Gesteland RF, Cech TR, Atkins JF (eds) (1999) The RNAworld. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY

3. Keenan RJ, Freymann DM, Stroud RM, Walter P (2001) AnnuRev Biochem 70:755–775

4. Latham JA, Cech TR (1989) Science 245:276–2825. Celander DW, Cech TR (1991) Science 251:401–4076. Cate JH, et al. (1996) Science 273:1678–16857. Golden BL, Gooding AR, Podell ER, Cech TR (1998) Science282:259–264

8. Theil EC, Eisenstein RS (2000) J Biol Chem 275:40659–406629. Gray NK, Hentze MW (1994) Mol Biol Rep 19:195–20010. Edwards TA, Pyle SE, Wharton RO, Aggarwal A (2001) Cell

105:281–28911. Manning GR (1978) Q Rev Biophys 11:179–12. Record MT (1978) Q Rev Biophys 11:103–13. Draper DE (1996) Nat Struct Biol 3:397–40014. Tinoco IJ, Bustamante C (1999) J Mol Biol 293:271–28115. Shiman R, Draper D (2000) J Mol Biol 302:79–9116. Treiber D, Williamson J (1999) Curr Opin Struct Biol 9:339–

34517. Fang X, Pan T, Sosnick T (1999) Nat Struct Biol 6:1091–

1095

18. Swisher JA, Su LJ, Brenowitz M, Anderson VE, Pyle AM(2002) J Mol Biol 315:297–310

19. Pan T, Long DM, Uhlenbeck OC (1993) In: Gesteland R,Atkins J (eds) The RNA world. Cold Spring Harbor Labora-tory Press, Cold Spring Harbor, NY, pp 271–302

20. Pyle AM, Green JB (1995) Curr Opin Struct Biol 5:303–31021. Basu S, et al. (1998) Nat Struct Biol 5:986–99222. Lorsch JR, Szostak JW (1994) Biochemistry 33:973–98223. Daniels D, Michels WJ, Pyle AM (1996) J Mol Biol 256:31–4924. Gonzalez RL, Tinoco IJ (2001) Methods Enzymol 338:421–44325. Pyle AM (1996) In: Sigel H, Sigel A (eds) Metal ions in bio-

logical systems, vol 32. Dekker, New York, pp 479–51926. Grosshans CA, Cech TR (1989) Biochemistry 28:6888–689427. Smith D, Burgin AB, Haas ES, Pace NR (1992) J Biol Chem

267:2429–243628. Hargittai MR, Musier-Forsyth K (2000) RNA 6:1672–168029. Ciesiolka J, Marciniec T, Krzyzosiak WJ (1989) Eur J Biochem

182:445–45030. Feig AL, Panek M, Horrocks WD, Uhlenbeck OC (1999) Chem

Biol 6:801–81031. Kieft JS, Tinoco I (1997) Structure 5:713–72132. Tinoco IJ, Kieft JS (1999) Nat Struct Biol 6:509–51133. Misra VK, Draper DE (1998) Biopolymers 48:113–13534. Misra VK, Draper DE (2000) J Mol Biol 299:813–82535. Rudisser S, Tinoco IJ (2000) J Mol Biol 295:1211–122336. Cate JH, Doudna JA (1996) Structure 4:1221–122937. Batey RT (2000) Science 287:1232–123938. Misra VK, Draper DE (2001) Proc Natl Acad Sci USA

23:12456–1246139. Cate JH, Hanna RL, Doudna JA (1999) Nat Struct Biol 6:553–

55840. Correll CC, Freeborn B, Moore PB, Steitz TA (1997) Cell

91:705–71241. Christian EC, Yarus M (1993) Biochemistry 32:4475–448042. Gordon PM, Piccirilli JA (2000) Nat Struct Biol 7:893–89843. Yoshida A, Sun S, Piccirilli JA (1999) Nat Struct Biol 6:318–32144. Chin K, Sharp K, Honig B, Pyle AM (1999) Nat Struct Biol

6:1055–106145. Rook M, Treiber D, Williamson J (1999) Proc Natl Acad Sci

USA 96:12471–46. Kuo LY, Piccirilli JA (2001) Biochim Biophys Acta 1532:158–

16647. Piccirilli JA, Vyle JS, Caruthers MH, Cech TR (1993) Nature

361:85–8848. Shan S, Kravchuk AV, Piccirilli JA, Herschlag D (2001) Bio-

chemistry 40:5161–517149. Shan SO, Herschlag D (2000) RNA 6:795–81350. Derrick WB, Greef CH, Caruthers MH, Uhlenbeck OC (2000)

Biochemistry 39:4947–495451. Brautigam CA, Steitz TA (1998) J Mol Biol 277:363–37752. Smith JS, Nikonowicz EP (2000) Biochemistry 39:5642–565253. Shan S, Yoshida A, Sun S, Piccirilli JA, Herschlag D (1999)

Proc Natl Acad Sci USA 96:12299–1230454. Beebe JA, Kurz JC, Fierke CA (1996) Biochemistry 35:10493–

1050555. Christian EL, Kaye NM, Harris ME (2000) RNA 6:511–51956. Gordon PM, Sontheimer EJ, Piccirilli JA (2000) Biochemistry

39:12939–1295257. Sontheimer EJ, Gordon PM, Piccirilli JA (1999) Genes Dev

13:1729–174158. Yean SL, Wuenschell G, Termini J, Lin RJ (2000) Nature

408:881–88459. Sontheimer EJ, Sun S, Piccirilli JA (1997) Nature 388:801–80560. McKay DB, Wedekind JE (1999) In: Gesteland RF, Cech TR,

Atkins JF (eds) The RNA world. Cold Spring Harbor Labo-ratory Press, Cold Spring Harbor, NY, pp 265–286

61. Takagi Y, Warashina M, Stec WJ, Yoshinara K, Taira K (2001)Nucleic Acids Res 29:1815–1834

62. Lilley DMJ (1999) Curr Opin Struct Biol 9:330–33863. Symons RH (1997) Nucleic Acids Res 25:2683–268964. Nakano S, Chadalavada DM, Bevilacqua PC (2000) Science

287:1493–1497

689

65. Murray JB, Seyhan AA, Walter NG, Burke JM, Scott WG(1998) Chem Biol 5:587–595

66. Walter N, Yang N, Burke J (2000) J Mol Biol 298:539–55567. Nesbitt SM, Erlacher HA, Fedor MJ (1999) J Mol Biol

286:1009–102468. O’Rear JL, Wang S, Feig AL, Beigelman L, Uhlenbeck OC,

Herschlag D (2001) RNA 7:537–54569. Lott WB, Pontius BW, von Hippel PH (1998) Proc Natl Acad

Sci USA 95:542–54770. Nesbitt S, Hegg LA, Fedor MJ (1997) Chem Biol 4:619–63071. Young KJ, Gill F, Grasby JA (1997) Nucleic Acids Res

25:3760–376672. Curtis EA, Bartel DP (2001) RNA 7:546–552.73. Perrotta AT, Shih I, Been MD (1999) Science 286:123–12674. Piccirilli JA, McConnell TS, Zaug AJ, Noller HF, Cech TR

(1992) Science 256:1420–142475. Noller HF, Hoffarth V, Zimniak L (1992) Science 256:1416–

141976. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA (2000) Sci-

ence 289:905–92077. Swisher JA, Duarte C, Su LJ, Pyle AM (2001) EMBO J

20:2051–206178. Moore MJ, Query CC, Sharp PA (1993) In: Gesteland R,

Atkins J (eds) The RNA world. Cold Spring Harbor Labora-tory Press, Cold Spring Harbor, NY, pp 303–357

79. Baserga SJ, Steitz JA (1993) In: Gesteland R, Atkins J (eds) TheRNA world. Cold Spring Harbor Laboratory Press, ColdSpring Harbor, NY, pp 359–382

80. Cech TR (1993) In: Gesteland R, Atkins J (eds) The RNAworld. Cold Spring Harbor Laboratory Press, Cold SpringHarbor, NY, pp 239–270

81. Collins CA, Guthrie C (2000) Nat Struct Biol 7:850–85482. Shukla GC, Padgett RA (2002) Mol Cell (in press)83. Pyle AM (1996) In: Eckstein F, Lilley DMJ (eds) Nucleic acids

and molecular biology, vol 10. Springer, Berlin Heidelberg NewYork, pp 75–107

84. Qin PZ, Pyle AM (1998) Curr Opin Struct Biol 8:301–30885. Sigel RKO, Vaidya A, Pyle AM (2000) Nat Struct Biol 7:1111–

111686. Qin PZ, Pyle AM (1997) Biochemistry 36:4718–473087. Jarrell KA, Dietrich RC, Perlman PS (1988) Mol Cell Biol

8:2361–236688. Peebles CL, Belcher SM, Zhang M, Dietrich RC, Perlman PC

(1993) J Biol Chem 268:11929–1193889. Abramovitz DL, Friedman RA, Pyle AM (1996) Science

271:1410–141390. Konforti BB, Abramovitz DL, Duarte CM, Karpeisky A,

Beigelman L, Pyle AM (1998) Mol Cell 1:433–44191. Zhang L, Doudna JA (2002) Science 295:2084–208892. Steitz TA, Steitz JA (1993) Proc Natl Acad Sci USA 90:6498–

650293. Woodson, et al. (1997) J Mol Biol 273:7–94. Woodson, et al. (2000) J Mol Biol 296:133–95. Tinoco I, Kieft J (1997) Nat Struct Biol 4:509–96. Chin et al. (1999) Nat Struct Biol 6:97 Feig AL (2002) The use of manganese as a probe for elucidating

the role of magnesium ions in ribozymes. In Metal Ions in Bi-ological Systems. vol 37: Manganese and its Role in BiologicalProcess (Sigel H, and Sigel A, Eds.) pp157–182, Marcel Dekker,New York

690