thomas r. cech and barbara l. golden howard hughes medical

The RNA World, Second Edition © 1999 Cold Spring Harbor Laboratory Press 0-87969-561-7/99 321

13Building a Catalytic Active Site Using Only RNA

TThhoommaass RR.. CCeecchh aanndd BBaarrbbaarraa LL.. GGoollddeennHoward Hughes Medical InstituteDepartment of Chemistry and BiochemistryUniversity of ColoradoBoulder, Colorado 80309-0215

WHERE DO RIBOZYMES FIT IN THE RNA WORLD HYPOTHESIS?

Two of the most fundamental requirements for life are information stor-age and catalytic function. Without storage, transfer, and replication ofinformation, a system cannot learn from its past and improve its viability;that is, there can be no natural selection. Equally essential to life is cat-alytic (enzymatic) function. At the very least, there must be machinery tocatalyze the copying of the informational molecules. This process mustproceed with considerable fidelity, yet some frequency of errors is alsonecessary to provide the diversity that allows adaptation and evolution.Beyond replication of the genome, additional catalytic functions would behighly advantageous to provide basic metabolism for even a primitiveself-reproducing system.

In contemporary organisms information is stored in the form of DNA.However, the persistence of RNA genomes in many viruses shows us thatthis sister nucleic acid is competent for information storage, at least forsmall genomes. Biocatalytic function in the modern world is mostly thedomain of protein enzymes, although ribonucleoproteins (RNPs) still cat-alyze the essential cellular reactions of protein synthesis and RNA splic-ing. By what evolutionary pathway did this DNA-RNA-protein solution tothe problem of life come about? The finding that RNA, an informationalmolecule, can by itself catalyze biochemical reactions has rekindledenthusiasm for the possibility that a key intermediate stage was an RNAWorld, with RNA providing both information and function, genotype andphenotype.

One version of this RNA World hypothesis is diagrammed in Figure 1.RNA is usually considered to be too complex a polymer to arise by

322 T.R. Cech and B.L. Golden

random chemistry, thus the proposal of a simpler progenitor (see Chapters2 and 6). The advantages of replicating a more homogeneous polymerwith a single type of building block may have driven the emergence of anRNA World, with RNA catalyzing its own replication (see Chapter 5). Atthis stage ribozymes, the subject of this chapter, would speed up and pro-vide specificity to biochemical reactions. Even at the very earliest stages,ribozymes would probably work in concert with whatever randomlyassembled peptides and other molecules were in their environment. Butonce a system stumbled upon a means to direct the synthesis of specificpeptide reproducibly—that is, to translate information from RNA to pro-tein (see Chapters 7 and 8)—ribozymes would start working as RNPenzymes. Thus, the RNA and RNP worlds would overlap extensively.Many of the ribozymes that function in contemporary cells continue towork as RNPs, as discussed in Chapters 14 and 18.

Will we ever know whether such an RNA World really existed?Perhaps not, but we can test the chemical plausibility of ribozymes per-forming diverse catalytic roles. Is RNA catalysis fast enough, specificenough, and versatile enough to support complex metabolism? How doescatalysis by RNA compare with that attainable by proteins, and what isthe structural basis of the similarities and differences? These are the ques-tions we address here.

RIBOZYME-CATALYZED REACTIONS

How Versatile is RNA Catalysis?

Natural ribozymes (Table 1) are all involved in RNA processing reac-tions. RNase P cleaves the 5� leader from primary transcripts of tRNAs.Chemically, this reaction is phosphodiester bond hydrolysis (Fig. 2a).

Figure 1 Role of ribozymes in an RNA World hypothesis.

Mechanisms of RNA Catalysis 323

Group I and II introns mediate the splicing of the RNA in which theyreside by a series of two concerted phosphodiester bond cleavage–ligation(transesterification) reactions (Fig. 2a). Group II RNAs also cleave andinsert themselves into double-stranded DNA in a reaction that requires thecooperation of the ribozyme active site and a protein moiety (Zimmerly etal. 1995). Small ribozymes, such as the hammerhead, clip RNA replica-tive intermediates to make unit-size progeny molecules by a differenttransesterification mechanism (Fig. 2b) (see Chapter 11). All these reac-tions are quite similar, in that the substrates are themselves nucleic acids,and chemically they all involve attack of a nucleophile at a phosphate.This is only a very small segment of the constellation of reactions cat-alyzed by protein enzymes.

Ribozymologists have reengineered self-splicing and self-cleavingribozymes to act as multiple-turnover catalysts by separating the portion

Table 1 Natural ribozymes

Number Biological Reaction performedCategory sequenced sources (reaction product)

Self-splicing RNAs transesterifica-Group I >500 Eukaryotes (nuclear tion (3�-OH)

and organellar), prokaryotes, bacteriophage

Group II >100 Eukaryotes (organellar),prokaryotes

Self-cleavingGroup I-like 6 Didymium, Naeglaria Hydrolysis

(3�-OH)

Small self-cleavers transesterifica-hammerheads 11 Plant viroids and satellite tion (2�, 3�>p)

RNAs, newthairpin 1 Satellite RNA of tobacco

ringspot virusHDV 2 Human hepatitis virusVS 1 Neurospora mitochondria

RNase P RNAs >100 Eukaryotes (nuclear Hydrolysis bacterial and organellar), (3�-OH)

prokaryotes

Selected references. Group I: Gott et al. 1986; Reinhold-Hurek and Shub 1992;Saldanha et al. 1993; http://pundit.icmb.utexas.edu. Group II: Cupertino and Hallick 1991; Michel and Ferat 1995. Self-cleaving group I-like: Einvik et al. 1997. Small self-cleavers: Symons, 1992. RNase P: http://jwbrown.mbio.ncsu.edu/ RNaseP/home.html; seeChapter 14.


of the molecule that contains the active site from the portion that under-goes reaction (Zaug and Cech 1986a; Zaug et al. 1986). This concept hasbeen extended to so many catalytic RNA systems that it is already com-monplace, yet it is critical for versatility: An “enzyme” restricted to a sin-gle round of reaction with a portion of itself would have extremely limitedroles in an RNA World.

Ribozymologists have also coaxed natural ribozymes to catalyzealternative chemistries. Group I and group II ribozymes can cleave single-stranded DNA as well as RNA (Herschlag and Cech 1990a; Robertsonand Joyce 1990; Mörl et al. 1992). The Tetrahymena ribozyme can trans-

Figure 2 Reactions catalyzed by ribozymes, both natural (a, b) and selected invitro (c–f ). (a) Hydrolysis or transesterification of a phosphodiester linkage (R= polynucleotide) or a phosphomonoester linkage (R = H). NOH = water forRNase P, NOH = 3�-OH of guanosine for group I introns, and NOH = 2�-OH ofan adenosine within the RNA for group II introns. (b) RNA cleavage by attack ofthe adjacent 2�-OH, the reaction catalyzed by the hammerhead and other smallribozymes. (c) RNA chain elongation using a nucleoside triphosphate substrate(R = H) or RNA ligation (R = polynucleotide). In either case, NOH = RNA chainwith a free 3�-OH (or 2�-OH). (d ) Amide-bond formation (Nu = 5�-NH2 of a mod-ified RNA), peptidyl transfer (Nu = NH2 group of another amino acid), or esterhydrolysis (Nu = water). In all cases, R is an amino acid side chain. (e) Diels-Alder cycloaddition. (f) Porphyrin metalation.


fer a 3�-terminal phosphate from one RNA strand to another, and it canhydrolyze a 3�-terminal phosphate (Zaug and Cech 1986b). These reac-tions involve phosphate monoester substrates instead of the usual diester.These DNA-cleavage and phosphomonoester reactions do not extend theribozyme repertoire greatly, as the substrates are still nucleic acid and thereactions still involve phosphorus centers. Although the Tetrahymenaribozyme has aminoacyl ester hydrolysis activity, a reaction that requiresattack of water at a carbon center (Fig. 2d), the amount of catalysis ismodest (Piccirilli et al. 1992).

The explosion of types of ribozyme-catalyzed reactions came with theadvent of in vitro selection/evolution technology, developed in the labora-tories of L. Gold, G. Joyce, and J. Szostak in 1990 (see Chapters 2, 5 and 6).In brief, a large combinatorial library of diverse RNA sequences is chal-lenged to perform some “task,” like catalyzing a specific reaction, and therare molecules that succeed are isolated and amplified to give a new pop-ulation, enriched in competent molecules. The beauty of the method is thathuge populations of sequences, such as 1015, can be sampled. In vitro evo-lution has led to the discovery of RNA and DNA molecules that utilizenucleoside triphosphate substrates (Fig. 2c) (Lorsch and Szostak 1994;Ekland and Bartel 1996); make and break amide bonds (Dai et al. 1995;Lohse and Szostak 1996; Wiegand et al. 1997), including an amide bondbetween two amino acids (Zhang and Cech 1997); alkylate a nucleoside ora thiophosphate (Wilson and Szostak 1995; Wecker et al. 1996), and addan amino acid to a nucleotide via an ester linkage (Illangasekare et al.1995). In all of these cases, the substrates still have an essential nucleicacid component, but the reactions involve carbon centers as well as phos-phorus centers. Thus, the spectrum of reactions that can be catalyzed byRNA is far greater than could be inferred from looking at natural ribozymes.

Two recent examples push the envelope even further. In vitro selec-tion has led to the discovery of modified RNA that catalyzes a classicorganic chemistry reaction, Diels-Alder cycloaddition, in the presence ofCu++ ions (Fig. 2e) (Tarasow et al. 1997). In addition, both RNA andDNA molecules have been selected to catalyze the insertion of metal ions(Cu++, Zn++) into porphyrin rings (Fig. 2f), similar to a step in the biosyn-thesis of heme (Conn et al. 1996; Li and Sen 1996). The former reactioninvolves carbon–carbon bond formation, whereas the latter requires a conformational change in the substrate but no covalent bond formation.The other breakthrough in these studies was that the substrates were notdirectly attached to a nucleic acid component, so interactions other thansubstrate–ribozyme base-pairing are responsible for positioning the sub-strate within the ribozyme active site.


How Fast is RNA Catalysis?

Catalytic rate can be assessed in numerous ways, only two of which arediscussed here. First, the turnover number of the enzyme (kcat) is infor-mative, because it gives the number of substrate molecules converted toproduct per minute by a single catalyst at saturating substrate concen-tration. Second, the rate constant for the chemical step of the catalyzedreaction can be compared to the rate constant for the same reaction uncat-alyzed. In more technical terms, the first-order rate constant for the reac-tion of one enzyme-bound substrate (this is simply kcat in those caseswhere the chemical step is rate-limiting overall) is divided by the first-order rate constant for the uncatalyzed reaction at the same temperature.Both of these parameters are listed in Table 2 for a few select ribozymesand for some protein enzymes that catalyze related reactions.

One main conclusion from Table 2 is ribozymes that evolved innature to catalyze reactions with nucleic acid substrates (e.g., group Iintrons) increase the intrinsic rate of a reaction by huge factors, within therange achieved by protein enzymes. Thus, RNA is not intrinsically a poorcatalyst compared to protein. On the other hand, ribozymes selected in thelaboratory to catalyze reactions with non-nucleic acid substrates havebeen measured to have more modest rate enhancements, no more than~1000-fold. It remains to be seen if this represents a fundamental inferi-

Table 2 Speed of ribozyme-catalyzed reactions

Rate enhancement Turnover numberkcat/kuncat

a kcat (min–1)

RibozymeGroup I, Tetrahymena 1011 0.1Group I, Anabaena 1010 4hammerhead 106 ≥1RNA ligase 109 100porphyrin metalation 460 0.9Diels-Adler 800 —

EnzymeT7 RNA polymerase 3 × 1011 14,000DNA ligase, E. coli 109 28

Values are taken from Herschlag and Cech 1990b, Zaug et al. 1994, Hertel et al. 1997,Ekland and Bartel 1995, Conn et al. 1996, Tarasow et al. 1997 and Bartel and Szostak1993.

a kcat refers to the chemical step of the reaction (where known), and therefore may notbe the same as kcat (turnover number). For the Diels-Adler reaction, rate enhancement isinstead based on ratio of second-order rate constants.


ority of ribozymes for such reactions, or a limitation of the in vitro selec-tion experiments.

Considering now the turnover numbers, even the ribozymes withimpressive catalytic rate enhancements are slow at processing substratesunder multiple turnover conditions. How is this possible? A reaction canonly proceed as fast as its slowest step. Ribozymes often bind nucleic acidsubstrates by Watson-Crick base-pairing (secondary structure) plus addi-tional “tertiary” interactions, which add up to give very tight binding. Thesame interactions stabilize binding of the nucleic acid reaction product,slowing its dissociation from the active site. Thus, rate-limiting productdissociation makes some ribozymes such as the Tetrahymena ribozymevery sluggish multiple-turnover catalysts (Herschlag and Cech 1990b;Young et al. 1991). However, this should not be considered a deficiency,because these catalytic introns have evolved as single turnover catalysts.If these RNAs had a rapid turnover rate, they would be able to react withother cellular RNAs once excised from their host RNAs.

How Specific Is an RNA Catalyst?

Protein enzymes are notable for their exquisite specificity, their ability todistinguish between very closely related substrates. How do ribozymescompare? Group I introns use guanosine (G) as a nucleophile to cleave the 5� splice site (Fig. 3), and other nucleosides have much reduced orundetectable activity (Bass and Cech 1984). When Michel et al. (1989b)located the G-binding site, they also identified specific hydrogen-bondinginteractions that explain the specificity (Fig. 4a). For the Tetrahymenaribozyme, the specificity for G over 2-aminopurine ribonucleoside isabout 1000-fold (Legault et al. 1992), whereas single base-pair changes inthe G-binding site reverse this specificity. An even higher specificity forG over deoxyG (Bass and Cech 1984) has been attributed to the favorableinteraction of a metal ion with the 2�-OH (Sjögren et al. 1997).

Another remarkable illustration of RNA’s potential to discriminatebetween closely related molecules is an in-vitro-selected aptamer thatbinds theophylline >10,000 times more tightly than caffeine, a moleculethat is identical except for one methyl group (Jenison et al. 1994). In thiscase, NMR structural analysis supports the model that substitution of theN7 proton with an N7 methyl group in caffeine would remove two Hbonds that the aptamer uses to bind theophylline and also disrupt stackinginteractions above and below the plane shown in Figure 4b (Zimmermannet al. 1997).


On the other hand, ribozymes that rely on base-pairing to bind nucleicacid substrates often have their specificity limited by the fact that mis-matches within nucleic acid helices are not very destabilizing (see Chapter10). Even worse, the slow rates of helix dissociation can give enough timefor cleavage to occur before a more weakly bound mismatched substratecan dissociate (Herschlag and Cech 1990c; Hertel et al. 1996). For exam-ple, the Tetrahymena ribozyme shows a 200-fold specificity for cleavageof the “matched” substrate GGCCCUCUAAAAA over the “mismatched”substrate GGCCCGCUAAAAA at low concentration of G nucleophilewhere the reaction is slow, but the specificity decreases to 4.5-fold at highG concentration (Herschlag and Cech 1990c). Mutant ribozymes that bindsubstrate less tightly do show increased sequence specificity (Young et al.

Figure 3 Group I intron structure and reactivity. (a) Expanded view of sec-ondary structure of a minimal group I intron. P = paired region. Thick lines rep-resent the RNA backbone. Thin lines show connectivity in this expanded viewbut in fact have zero length. Shaded regions = catalytic core. In the first step ofself-splicing, exogenous guanosine or GTP binds in the G-site and cleaves the 5�

splice site (large arrowhead). The 3�-terminal (ω) guanosine of the intron (cir-cled) then occupies the G-site for the second step of splicing. (b) Self-splicing.First step, 5� splice-site cleavage by exogenous guanosine. Second step, 3� splice-site cleavage by the 5� exon at the bond following ωG (circled ), resulting in exonligation.


1991). The hammerhead ribozyme also has some ability to distinguishmismatches in a ribozyme-substrate helix away from the catalytic core,but it has dramatic discrimination against mismatches that flank the core(Werner and Uhlenbeck 1995; Hertel et al. 1997). Presumably, theseproximal mismatches destabilize the catalytically active structure. Thus,there are cases in which a ribozyme recognizes a nucleic acid substratewith more specificity than that intrinsic to base-pairing interactions. Such

Figure 4 Specific binding of small molecules by RNA. (a) Guanosine bindingby a conserved G-C base pair in the P7 duplex of group I introns, and change ofspecificity (G → 2-aminopurine ribonucleoside) upon switching to an A-U basepair in P7 (Michel et al. 1989b). (b) Theophylline-binding site in an in-vitro-selected aptamer. The circled H is substituted by a methyl group in caffeine, dis-rupting binding (Zimmermann et al. 1997).


a feature seems critical to obtaining sufficient fidelity for RNA-catalyzedRNA replication in an RNA World.

Ribozymes discriminate easily between stereoisomers of the same sub-strate, as expected for any catalyst that provides a chiral three-dimensionalactive site. A dramatic example is provided by the Tetrahymena ribozyme,which cleaves the RP and SP diastereomers of an RNA substrate with a>1000-fold different rate, even though these differ only in the position ofa subtle oxygen-to-sulfur substitution (Cech et al. 1992). As anotherexample, an RNA selected for binding to D-tryptophan agarose bound>700 times more weakly to L-tryptophan agarose (Famulok and Szostak1992). An exciting application of this stereospecificity is “mirror image”drug development: One can select a nucleic acid aptamer that binds to themirror image (enantiomer) of a target protein, and then be assured that theenantiomer of the selected aptamer will be specific for the naturally occur-ring isomer of the target protein (Klussmann et al. 1996; Williams et al.1997). The enantiomer of the aptamer, composed of unnatural L-ribosesugars, is not recognized by natural nucleases and therefore has the poten-tial to be a long-lived drug in vivo.

MECHANISMS OF RNA CATALYSIS

How do ribozymes lower the activation energy and thereby speed up bio-chemical reactions? The much-studied group I ribozymes will be used toillustrate the catalytic strategies available to RNA, with other systemsbeing compared and contrasted along the way (see also Narlikar andHerschlag 1997).

Binding and Orienting Reactive Groups

When enzymes bind substrates, they create a high local concentration andoptimal orientation to increase the efficiency of reaction (Jencks 1969).Group I introns use this same strategy, binding guanosine in proximity tothe 5� splice site and converting what would normally be an intermolecu-lar reaction into an effectively intramolecular reaction (Bass and Cech1984). Guanosine binding is entropically driven, perhaps indicative ofrelease of solvent from the RNA upon G-binding (McConnell and Cech1995). Elements of the ribozyme that contribute to G-binding (Fig. 4a)(Michel et al. 1989b; Yarus et al. 1991) and to binding of the 5�-splice sitehave been identified. (The latter set of interactions is described below inBuilding a Catalytic Center Using Only RNA, because it provides a goodgeneral model for specific helix-packing interactions in large RNAs.)


Although we know that these two reactants are bound in a manner thatallows them to react, there is no information about whether they are heldin anything close to an optimal orientation.

Group II introns, found in certain mitochondria, chloroplasts, andbacteria, have conserved structure distinct from that of group I (Michel etal. 1989a). Group II introns also accomplish self-splicing by consecutivetransesterification reactions, but with a twist: In the first step, the 2�-OHof a specific intronic adenosine (A) cleaves the 5� splice site to form a“lariat” intermediate, and in step two, the cleaved 5� exon attacks the 3�-splice site, ligating the surrounding exons (Peebles et al. 1986; Schmelzerand Schweyen 1986; van der Veen et al. 1986). In the laboratory, group IIintrons can catalyze trans reactions, such as binding a free 5�-exon analogand ligating it to the 3�-exon (Jacquier and Rosbash 1986). Both the cisand trans reaction rely on two exon-binding sites that base-pair with complementary 5�-exon sequences (intron binding sites or IBS, Fig. 5;Jacquier and Michel 1987). These long-range interactions are similar tothe internal guide sequence-5�-exon pairing of group I introns (Davies et al.1982; Michel et al. 1982). In addition, a portion of the conserved GUGYGsequence at the 5� end of group II introns pairs with a loop within domainI of the intron, further contributing to 5�-splice site recognition (Jacquierand Michel 1990). Presumably, additional, as-yet-unidentified tertiaryinteractions position the 2�-OH of the nucleophilic A in domain VI forattack.

Step 2 of group II intron self-splicing requires an alternate structuralinteraction (Chanfreau and Jacquier 1996; η/η� in Fig. 5). This is espe-cially noteworthy, because although there is much evidence for confor-mational changes in ribozyme catalysis (see, e.g., Been and Perrotta 1991;Wang et al. 1993; Golden and Cech 1996), there are few examples wherea specific structure has been identified that orchestrates a conformationalswitch. In the group II intron, the branch-site A for step 1 of splicing mustbe displaced from the active site to allow entry of the last intronnucleotide and the 3�-splice site (Steitz and Steitz 1993; Jacquier 1996).Branch formation or 5�-splice site cleavage may result in a displacementthat allows the η/η� base-pairing to form, and its formation may in turnpull the branched A from the active site, allowing the last intronnucleotide to enter for step 2 of splicing.

Metal Ion Catalysis

Activity of most ribozymes requires, or is greatly stimulated by, divalentmetal ions such as Mg++, Mn++, and in some cases Ca++. These could be


involved in folding the polyanionic RNA catalyst, in active-site chem-istry, or in both. Plausible chemical roles have been much discussed pre-viously (Pyle 1993; Yarus 1993) and include

1. electrostatic stabilization of an anionic attacking or leaving group2. electrostatic stabilization of an electrophilic group (Lewis acid

catalysis)3. electrostatic destabilization of a substrate in the ground state4. specific orientation of substrates by metal ion coordination5. acid-base catalysis by metal-bound water or hydroxide6. oxidation-reduction chemistry

In favorable cases, the structural and catalytic roles of metal ions havebeen separated. The Tetrahymena ribozyme requires divalent metal ionsin order to attain its active three-dimensional folded structure (Latham andCech 1989; Celander and Cech 1991), a requirement that can be met by a

Figure 5 Group II intron structure. Intron-binding sites (IBS 1 and 2) located inthe 5� exon are base-paired to two intronic exon-binding sites (EBS 1 and 2,respectively). Greek letters designate other tertiary interactions; those discussedin the text are connected by dashed lines. The first step of self-splicing, attack ofthe 2�-OH of an A in domain VI at the 5� splice site, is indicated by an openarrow.


variety of divalent cations and partially met by high concentrations ofmonovalent cation (Downs and Cech 1996). In addition, correct position-ing of the 5� splice site helix within its active site has a requirement forMg++ that cannot be met by Ca++ (Wang and Cech 1994; McConnell et al.1997).

Because numerous metal ions are required to fold the ribozyme, it wasdifficult to identify additional metal ions involved directly in active-sitechemistry, especially since they are already bound at the 2 mM Mg++ con-centration required for ribozyme folding (McConnell et al. 1997). Thisproblem was addressed by site-specific substitution of atoms that wouldpredictably perturb the binding and function of a coordinated metal ion.In the Tetrahymena ribozyme, substitution of bridging oxygen atoms bysulfur at the reactive bonds led to loss of activity with Mg++. Restorationof cleavage with thiophilic metal ions such as Mn++ and Cd++ in both theforward and reverse reactions led to the proposal of metal ions 1 and 2 inFigure 6. Metal ion 1 destabilizes the substrate in the ground state but sta-bilizes the developing negative charge as the O–P bond is broken in thetransition state, thereby providing roughly 106-fold catalysis (Piccirilli etal. 1993; Narlikar et al. 1995). Metal ion 2 acts to deprotonate the 3�-OHof the G nucleophile (Weinstein et al. 1997). These two metal ions mayform the sort of “two metal ion center” proposed by Steitz and Steitz(1993), a catalytic strategy with clear connections to the world of proteinenzymes (Beese and Steitz 1991; Kim and Wyckoff 1991). Substitution ofthe 2�-OH of the G nucleophile by an amino group also led to a metalspecificity switch, leading to the proposal of metal ion 3 (Fig. 6) (Sjögrenet al. 1997). Metal ion 3 could function to position or activate the nucle-ophile.

Divalent metal ions are required for the hammerhead ribozyme reac-tion under physiological conditions. There is evidence for their binding tothe reaction-site phosphate, enhancing its ability to be attacked by theoxyanionic 2� nucleophile, and also evidence for metal ion activation ofthe 2�-OH nucleophile (see Chapter 11). However, the hairpin ribozyme,which cleaves RNA by the same 2�, 3�-cyclic phosphate mechanism as thehammerhead, appears to use metal ions just for folding, not for chemistry.Cobalt hexammine, a metal complex inert to changes in its coordinationsphere, supports full hairpin ribozyme cleavage, providing evidenceagainst the importance of direct coordination of the metal to the RNA.Also unlike the hammerhead, phosphorothioate substitution at either non-bridging oxygen has small and metal-ion-independent effects on activity,evidence against any direct metal coordination (Hampel and Cowan 1997;Nesbitt et al. 1997; Young et al. 1997). This system serves as an impor-


tant reminder that RNA enzymes, like protein enzymes, will embracediverse catalytic strategies.

Covalent Catalysis

Many protein enzymes use a nucleophilic amino acid (e.g., serine, lysine,cysteine, or histidine) to attack a substrate, forming a covalent intermedi-ate with a portion of the substrate. In a second step, attack of the covalentintermediate by an external nucleophile (such as water) releases the prod-uct and restores the enzyme (e.g., serine proteases, or alkaline phos-phatase in Fig. 7).

The Tetrahymena ribozyme performs reactions that are highly analo-gous to those carried out by Escherichia coli alkaline phosphatase (Fig. 7).

Figure 6 Transition state stabilization by the Tetrahymena group I ribozyme.In-line attack of the 3�-OH of guanosine at the phosphorus atom equivalent to the5� splice site is supported by the stereochemical course of the reaction(McSwiggen and Cech 1989; Rajagopal et al. 1989). Metal ion catalysis (siteslabeled 1, 2, and 3) is discussed in the text.


Because the biological function of this RNA involves self-splicing reac-tions at phosphodiesters, it was not obvious that it should have reactivitywith phosphomonoester substrates, since the two have very different tran-sition states (Zaug and Cech 1986b). Nevertheless, the ribozyme effi-ciently transfers a 3�-terminal phosphate from an oligonucleotide to itsown 3�-terminal guanosine, forming a covalent E-p intermediate. Thephosphate can then be transferred to a different oligonucleotide (one thatcan also bind in the active site) or it can be hydrolyzed, the latter reactionbeing enhanced at low pH. In either case, the free catalyst E is restored,ready for another catalytic cycle.

Why is covalent catalysis advantageous? It allows an active site to beoccupied successively by two different substrates; in the example givenabove, the Tetrahymena ribozyme first binds the oligo CCCUCUp, storesthe phosphate as an E-p complex, binds a second oligo UCU, and trans-

Figure 7 Covalent catalysis by the Tetrahymena group I ribozyme. (a) Reactionof the E. coli protein enzyme alkaline phosphatase (E) involves binding of a phos-phorylated substrate (S) to form a noncovalent E·S complex, followed by trans-fer of the phosphate to an active-site serine to form a covalent E-P intermediate.Hydrolysis then restores the active enzyme. (b) The ribozyme has analogousactivity, with the 3�-terminal G of the nucleic acid acting like the serine of alka-line phosphatase.


fers the phosphate to it to form UCUp (Zaug and Cech 1986b). More gen-erally, the immobilization gained by forming a covalent intermediate maybe advantageous for catalysis.

Contemporary protein enzymes use covalent catalysis to act as pro-teases, esterases, phosphoglucomutases, transphosphorylases, and ligases,often with a serine OH group as a nucleophile. In an RNA World, it wouldbe feasible for ribozymes to perform similar reactions using a ribose 2�-or 3�-OH group as a nucleophile.

General Acid–Base Catalysis

Many chemical reactions require transfer of a proton to or from a reactant.The proximity of a proton donating or accepting group, that is, a generalacid or base, can greatly speed such reactions. RNA does not have histi-dine, but functional groups on the RNA bases or backbone could perhapsserve as proton donors or acceptors (Pace and Marsh 1985). However,with the exception of the 5�-terminal phosphate and modified nucleotidessuch as 7-methylguanosine, the pKa values of these groups are normallyfar from pH 7. Could an RNA structure perturb a pKa in an active site toprovide efficient general acid–base catalysis? In the absence of a con-firmed example, it seems possible that ribozymes will in general use metalions and metal-ion-bound solvent in lieu of general acid–base catalysis(Cech et al. 1992).

A DNAzyme that catalyzes the insertion of metal ions into mesopor-phyrin IX works by enhancing the basicity of the bound mesoporphyrinsubstrate by 3–4 pH units, thereby enhancing its ease of metallation (Liand Sen 1998). This may occur by distortion of the planar mesoporphyrinstructure by the DNAzyme, although involvement of a catalytic groupsuch as a negatively charged phosphate is another possibility.

Use of Binding Energy away from the Site of Reaction

To decrease the energy barrier for a reaction, an enzyme must stabilize thereaction’s transition state more than it stabilizes the bound substrate(s) inthe ground state. One strategy for doing so is to use binding energy awayfrom the site of reaction to “force” an interaction at the reaction site, aninteraction that induces the substrate to react (Jencks 1975). The enzymecan force entropic fixation of a substrate with respect to another substrateor with respect to active-site groups, or force electrostatic destabilizationby juxtaposing a (partially) charged substrate atom with a like charge onthe enzyme. In either case, if the strained interaction is relieved upon


approach to the transition state, catalysis will result. When enzymes usethis trick, the observed binding of the substrate(s) is weaker than it wouldotherwise be, because part of the intrinsic binding energy is used to paythe price of forcing the unfavorable ground-state interaction.

Recent studies with the Tetrahymena ribozyme have shown that thissophisticated strategy is used by RNA as well as protein enzymes. TheTetrahymena ribozyme binds its oligonucleotide substrate (CCCUCUpA)and product (CCCUCU-OH) by base-pairing with the CCCUCU portionplus additional “tertiary” interactions. The tertiary interactions are muchweaker with the substrate than with the reaction product, a difference due to the phosphate rather than the 3�-A residue (Narlikar et al. 1995).The authors suggest that the ribozyme uses binding interactions to forcethe bridging oxygen atom, partially positive in the ground state, next tothe positively charged magnesium ion 1 (Fig. 6); this substrate destabi-lization is relieved in the transition state as negative charge accumulateson the oxygen atom. They estimate 280-fold rate enhancement from theground state destabilization and another 60-fold from the additional posi-tive interactions in the transition state, for a combined catalytic effect thataccounts for 104-fold of the total 1011-fold catalysis achieved by thisribozyme (Narlikar et al. 1995).

BUILDING A CATALYTIC CENTER USING ONLY RNA

Solvent-inaccessible Core

Protein enzymes form a densely packed, hydrophobic core to support aconcave active site. Given the absence of hydrophobic side chains and thepolyanionic backbone of nucleic acids, how is it possible for them to forma functionally equivalent structure? Single-stranded nucleic acids certainlyfold into more compact structures than their double-helical counterparts.Transfer RNA and guanine quadruplexes provide well-studied examples,but even the tRNA structure does not provide much solvent inaccessibili-ty to its backbone; most of the backbone is still on the outside of themolecule, bathed in solvent, very different from a folded protein.

Large ribozymes, such as the Tetrahymena group I intron, do form arelatively solvent-inaccessible core as judged by protection from free-radical bombardment (Latham and Cech 1989; Celander and Cech 1991;Sclavi et al. 1997). This suggested that nucleic acid building blocks canbe packed together with the help of divalent cations to form somethingroughly equivalent to the core of a globular protein. In the following sec-tion, we explore the types of interactions that contribute to formation ofsuch a structure.


Interactions that Stabilize a Close-packed RNA Core

The crystal structure of one domain of the Tetrahymena ribozyme at 2.6 Å resolution provided the first atomic view of an RNA large enoughto have a relatively solvent-inaccessible core (Cate et al. 1996). This 160-nucleotide domain, called P4-P6, comprises about half of the active site ofthe ribozyme. When synthesized as a separate RNA molecule, it assumesits native secondary structure as well as a higher order tertiary structurethat appears to be a simple subset of its structure within the context of thewhole, active ribozyme (Murphy and Cech 1993, 1994). Examination ofthe structure (Fig. 8a) reveals both the expected nucleic acid features and

Figure 8 An RNA domain. (a) The P4-P6 domain of the Tetrahymena group Iintron rendered as CPK atoms, showing the side-by-side arrangement of two heli-cal subdomains. The two major sites of interaction between the two subdomainsare highlighted: the junction of P4 and P6 (blue), the A-rich bulge (red), theGAAA tetraloop (green), and the tetraloop receptor (violet). (b) Coordination oftwo magnesium ions (violet) by the backbone of the A-rich bulge displays thenucleotide bases for tertiary interactions with the rest of the domain. (c) TheGAAA tetraloop-receptor is stabilized both by stacking (highlighted in red ) andspecific hydrogen bonds between the two modules. For orientation purposes, theG of the GAAA tetraloop is drawn in blue.


also features that appear protein-like, at least superficially. The moleculehas a sharp bend at the top, which allows two punctuated double-helicalregions to be aligned side by side. The protein-like feature is the tightlypacked core produced by sandwiching together the two halves of themolecule. Thus, the P4-P6 structure provides the first opportunity to eval-uate the molecular basis for formation of such a higher order RNA struc-ture, as discussed below.

Long-range Base Pairs and Triples

The two halves of the P4-P6 RNA domain are brought together mainlythrough two tertiary interactions: The A-rich bulge docks into the minorgroove of P4 (Flor et al. 1989) and also into the P5abc three-helix junc-tion (Fig. 8b), whereas the L5b tetraloop is bound by a receptor sequence(Costa and Michel 1995) located between P6a and P6b in the other half ofthe molecule (Fig. 8c). Each of these interactions involves base triplesbetween a base pair within a duplex region and a third base that is faraway in the secondary structure of the molecule. However, thinking ofthese as simply base triples oversimplifies the interactions, as they involvemultiple H–bonds between 2�-hydroxyl groups and phosphate oxygens,and also extensive base-stacking interactions (Cate et al. 1996).

A similar lesson about base triples can be gleaned from an inter-molecular tetraloop/minor groove interaction seen in the crystal structureof the hammerhead ribozyme (Pley et al. 1994). Again base triples pro-vide the specificity of the interaction (often because substitution of a dif-ferent base would cause a steric clash), but a larger number of long-rangeH–bonds involve ribose hydroxyls and phosphate oxygen atoms.

Comparative sequence analysis and site-specific mutagenesis studieshave identified base triples and long-range base pairs in many otherribozymes whose detailed structures have not yet been solved. Examplesin group I ribozymes include the triple-helical scaffold involving P4 andP6 (Michel and Westhof 1990; Michel et al. 1990), base-pairing betweenperipheral loops that stabilize the sunY and Tetrahymena introns (Michelet al. 1992; Jaeger et al. 1993; Lehnert et al. 1996), and a long-range triplebetween P4 and J8/7 that brings together the domains of the Tetrahymenaand cyanobacterial introns (Tanner and Cech 1997). Examples in group IIribozymes so far include long-range base pairs between two hairpin loopsor between a hairpin loop and an internal loop (Michel et al. 1989a) and aGAAA tetraloop-receptor interaction that joins domains I and V (Costaand Michel 1995), as summarized in Figure 5. RNase P RNAs are com-pacted by long-range pseudoknots, one of which involves a hairpin loop (James et al. 1988; Haas et al. 1991), and other interactions (see


Chapter 14). As in the interactions seen in crystal structures, it seems likelythat each of these biochemically verified tertiary interactions will be but-tressed by a network of additional H–bonds involving the RNA backbone.

The 2�-Hydroxyl Group

The 2�-OH of the ribose sugar, the group that distinguishes RNA fromDNA, is exploited frequently for structural interactions in large RNAs.This should be expected, because once RNA engages in base-paired sec-ondary structure, its backbone is its most available element, and the 2�-OHgroups lining the minor groove are particularly available for higher-orderinteractions. As detailed above, H-bonds involving 2�-OH groups buttressbase triple interactions. In addition, helical regions can be held togetherby ribose zippers—the ribose and attached base of one nucleotide donateand accept H-bonds from the same 2�-OH of a second nucleotide, and thisinteraction is repeated at the adjacent level in a “ladder” of interactions.

Ribose 2�-hydroxyls also have a key role in 5�-splice-site recognitionin group I introns. This system is of general interest because it provides amodel for specific recognition of base-paired helices within folded RNAmolecules. The last few nucleotides of the 5�-exon are first recognized bybase-pairing to an intronic internal guide sequence (IGS; Davies et al.1982; Michel et al. 1982). The resulting duplex is called P1 (Fig. 3a). Ina kinetically separable step, this P1 helix is then positioned within theribozyme active site (Bevilacqua et al. 1992; Herschlag 1992). As shownin the model in Figure 9, certain 2�-OH groups on both strands of P1 arerecognized by the intron core (see, e.g., Pyle et al. 1992; Strobel and Cech1993; Strobel et al. 1998). Additionally, the exocyclic amino group of theG·U wobble base pair at the cleavage site is also important for splice-siterecognition and specificity (see, e.g., Strobel and Cech 1995). This G·Uwobble pair is conserved among most of the 500 known group I introns,and the two that have instead a G-C become more reactive when a G·U issubstituted at this position (Hur and Waring 1995). The docking of the P1helix within the group I active site illustrates how backbone and minorgroove interactions can be used to recognize an RNA element that has itsbase sequence information “hidden” in a helix.

In group II self-splicing introns (Fig. 5), domain V is a phylogenicallyconserved element that is essential for catalysis (Jarrell et al. 1988). As inthe P1–group I intron case, specific 2�-OH groups on one side of the helixmediate binding of domain V to the remainder of the intron (Abramovitzet al. 1996). On the opposite (major groove) face of the domain V helix,specific 2�-OH groups, phosphates, and bases (including the G of a G·U


pair) participate in transition-state stabilization and therefore appear toform part of the active site for 5� splice-site cleavage (Chanfreau andJacquier 1994; Peebles et al. 1995; Abramovitz et al. 1996).

Metal Ions

The role of divalent metal ions in stabilizing sites of closebackbone–backbone interaction has been studied in transfer RNA, boththermodynamically (Cole et al. 1972) and structurally (see Chapter 12).However, tRNA only provided details for the shorter-range interactions,not those which mediate helix packing on globular domains. The crystalstructure of the P4-P6 ribozyme domain revealed metal-ion-mediatedlong-range interactions. The close packing of helices brings their phos-phates into proximity; the resulting electrostatic repulsion is ameliorated

Figure 9 Substrate recognition by a group I ribozyme. An oligonucleotide sub-strate (S) is first recognized by base-pairing to a short complementary sequencecalled the internal guide sequence (IGS), forming the helix P1. The cleavage siteis defined by a G·U wobble pair at the top of the helix. The P1 helix then docksinto the active site of the intron (including elements P3, P4, P5, P7, P8, J4/5, andJ8/7). It is correctly positioned by interactions with 2�-OH groups (circled) andthe exocyclic amine group of the G·U wobble at the cleavage site. (Figure tem-plate courtesy of Scott Strobel.)


by binding of hydrated magnesium ions. These outer-sphere magnesium-ion complexes bridge P5 and P5a as well as P5b and P6a (Cate et al.1996). The crystal structure brought atomic detail to a picture that hadbeen inferred from studies of ribozyme folding as a function of Mg++ con-centration (Celander and Cech 1991; Banerjee et al. 1993; Jaeger et al.1993; Murphy and Cech 1994; Szewczak and Cech 1997).

A striking role of divalent cations is seen in the A-rich bulge of P4-P6.The backbone wraps around two divalents, each of which makes an inner-sphere coordination complex with three phosphate oxygens (Cate et al.1996). As a result, the backbone is on the inside of this local structure andthe bases protrude to make tertiary interactions with distant portions of themolecule (Fig. 8b). This structure provides an answer to the paradox ofSigler (1975), who pointed out that proteins form sensible secondarystructure, in that the side chains protrude to allow higher-order interac-tions, whereas nucleic acids form helices that tuck the side chains (bases)inside, not optimal for higher-order interactions. In this A-rich bulge, themetal ions turn a portion of the RNA “inside out,” constraining the back-bone to be inside and displaying the adenosine “side chains” for tertiaryinteractions. Phosphorothioate substitution at the metal-coordinating sitesinterferes both with local folding and with folding of the P4-P6 domain,confirming the essentiality of this metal binding (Cate et al. 1997).

In going from the isolated P4-P6 domain to the whole ribozyme,much more surface area is buried. Therefore, metal ions are likely to beeven more important. Consistent with this idea, a long string of function-ally significant metal-binding sites in the ribozyme core has been mappedby Christian and Yarus (1993) using phosphorothioate interference andMn++ rescue.

In summary, both inner-sphere and outer-sphere coordination of RNAfunctional groups by divalent cations contribute to the specificity and sta-bility of RNA tertiary structure.

Small Ribozymes Are Different

At this point we must introduce a disclaimer. We have been developing apicture of a globular RNA with a concave active site that maintains a sim-ilar form with or without substrates bound. That this is a reasonable modelfor the Tetrahymena ribozyme is supported by the small amount of ener-getic coupling between the binding of the guanosine substrate and the P1helix, indicative of a site that is only slightly reorganized upon bindingeither single substrate (Bevilacqua et al. 1993; McConnell et al. 1993).Similarly, chemical modification or cleavage studies have seen only


minor differences between the free ribozyme and substrate-bound ver-sions. However, small ribozymes such as the hammerhead are much dif-ferent. Here the binding of substrate is necessary for formation of the cat-alytic center (Hertel et al. 1997), and even after the substrate–ribozymecomplex is formed, the catalytically active conformation may be formedonly occasionally (see Chapter 11). Thus, the small ribozymes that wereuseful in the early stages of the RNA World were probably relativelyfloppy, and only after RNA self-replication became efficient enough tocopy a larger RNA (>100 nucleotides) could ribozymes with permanentactive sites come on the scene.

Ribozyme Dynamics

An atomic-resolution crystal structure or NMR structure of a macro-molecular catalyst is the single most valuable set of structural informationone can obtain. It provides a framework for planning and interpretingexperiments that probe the relationships between structure and catalyticfunction. Yet it gives largely a static picture, and even if a completelyrigid macromolecule could bind its substrates, it could never promotetheir reaction. Formation of a transition state requires movement. Onetype of movement—switches between distinct conformers—has beendescribed above. We now consider another type of dynamics—thermalmotions. These can be thought of as the excursions that a molecule under-goes from its most stable ground-state structure.

The magnitudes of thermal motions between two domains (the P1substrate domain and the P3-P9 domain) of the Tetrahymena ribozymehave been investigated by disulfide cross-linking (Cohen and Cech 1997).Reactive groups were appended to the RNA backbone by an adaptation ofthe 2�-modification method pioneered by Sigurdsson et al. (1995). Uponcollision, these groups can form a disulfide cross-link. Sites separated by50 Å in the structure were found to be cross-linked at rates only 3 to 15times slower than the rates for proximal sites, indicative of an unexpect-edly high degree of flexibility. The motions were estimated to occur onthe microsecond time scale at 30˚C (Cohen and Cech 1997).

Presumably, there will be fewer degrees of freedom and therefore lessflexibility within a single RNA domain than between two domains.Nevertheless, these measurements suggest that large, globular RNAs aremuch more dynamic than proteins. It remains to be tested whether one ofthe functions of RNA-binding proteins is to damp out these large-scalemotions, thereby conferring a selective advantage to the RNP World overthe RNA World (Fig. 1).


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thomas r. cech and barbara l. golden howard hughes medical

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