catalytic rna · otherrnaoligonucleotidesformingpseudoknots,kissing loopsormultistrandedhelices....

Catalytic RNAFrank Walter, Institut de Biologie Moleculaire et Cellulaire du CNRS, Strasbourg, France

Eric Westhof, Institut de Biologie Moleculaire et Cellulaire du CNRS, Strasbourg, France

Some RNA molecules can function like enzymes and exert a catalytic action on themselves

or on other molecules. These ribozymes are diverse in size and sequence and differ in

important aspects of their three-dimensional structure and folding. Catalytic RNAs present

a new target for drugs and can be used for inactivating unwanted RNA or DNA molecules

by a specific cleavage reaction.

Discovery

Until recently, protein enzymeswere thought to be the onlybiologically active catalysts. It was believed that DNAstored the genetic information and that RNA played therole of an intermediate courier between the geneticmessages contained in the DNA and the ribosomes, whereproteins are synthesized. Other RNAs, like transfer RNAs(tRNAs) and ribosomal RNAs (rRNAs), were consideredas helper molecules to assist the function of proteins. Thediscovery in the 1980s of catalytic RNAs revolutionizedmolecular biology (Cech et al., 1981; Guerrier-Takadaet al., 1983). Today,RNAmolecules are the onlymoleculesknown both to store genetic information (as in the RNAviruses and viroids or during transport in the form ofmRNA) and to exert biological catalysis.Catalytic RNAs, or ribozymes, are found widely in

nature and therefore have a nondispensable biologicalactivity. In the tRNA-processing enzyme ribonuclease P(RNase P), theRNAmoiety contains the catalytic activity.Self-splicing introns often occurwithin ribosomal genes, asin the rRNA of Tetrahymena thermophila. The humanhepatitis delta virusRNAand some plant virusRNAs, likethe tobacco ringspot virus satellite RNAs, require RNAself-cleavage and ligation for replication.The discovery of catalytic RNAs opened new vistas on

the evolution of RNA and the origins of life. It is nowtantalizing to imagine anRNAworld in whichRNA couldstore information for self-reproduction and self-processingwithout the need of proteins. Very recently, it has beenestablished that protein synthesis on the ribosome iscatalysed by the 23S ribosomal RNA compound and,thus, that the ribosome is actually a ribozyme (Nissen et al.,2000).Ribozymes need to acquire three-dimensional architec-

tures to promote specific interactions with cofactors,especially divalent metal ions, and other functionaldomains for processing RNA substrates. Ribozymes aregenerally built upof several structural subdomainsmadeofhelical segments connected by tertiary contacts. Func-tional regions are usually located in single-strandedregions, such as internal loops or bulges. Specific tertiary

contacts occur between hairpin and internal loops,especially those positioned on the outside of the molecule.The subdomains have various functions and are respon-sible for substrate recognition, specific sequence alignmentand catalytic activity, leading to a modular and hierarchi-cally organized architecture.Some ribozymes, like the hammerhead ribozyme, the

hairpin ribozyme and the RNAase P RNA, are underextensive clinical research for their ability to cleave otherspecifically chosen substrate RNAs. The therapeuticapplications range from cleavage of viral RNAs, like theacquired immune deficiency syndrome (AIDS)-causinghuman immunodeficiency virus (HIV) RNA, to silencingof carcinogenic ormutated cellularRNAs, or the control ofgene expression in vivo.

Sequence and Structure

RNAmolecules are built up of RNA helices interlinked byinternal loops and multiple junctions. Double-strandedhelical regions are formed through standard Watson–Crick base pairs. One groove of the standard type A-formdouble-stranded RNA helix is deep and narrow, while theother one is shallow and wide. Double-stranded regionsmainly act as a framework or rigid spacer to organize andto orientate other structural and functional recognitionelements. Helices can be connected by three-, four-ormultiway junctions, which form a sequence-dependentjoint, which is either flexible or fixed, depending, amongother factors, on the type and concentration of metal ions.Single-stranded regions comprise a whole range of

structural elements, including base bulges, internal loopsor bubbles and terminal or hairpin loops. These can simplyserve as flexible linkers between helical domains, or theyinteract with other bases, usually forming non-Watson–Crick pairs to widen or tighten the grooves or to inducekinks and bends in the RNA backbone. These regions alsoform tertiary interactions with other regions of the same or

Article Contents

Secondary article

. Discovery

. Sequence and Structure

. Catalytic Mechanisms

. Classes of Catalytic RNAs

. Intron Splicing

. Small Catalytic RNAs

. Artificial Ribozymes

. Interaction with Antibiotics

. Therapeutic Use of Ribozymes

. Final Remarks

1ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

otherRNAoligonucleotides forming pseudoknots, kissingloops or multistranded helices.

Catalytic Mechanisms

The best-studied natural RNA enzyme is the RNAasePRNA,which involves a site-specific hydrolysis (Figure1a).Other biologically occurring ribozymes catalyse anintramolecular or cis-reaction and are usually modified

during this reaction (Figure 1b–d). Therefore, they usuallycatalyse single turnover reactions in sequential steps.Apartfrom self-cleavage in an intramolecular cleavage or cis-reaction, ribozymes can also be engineered to act inintermolecular cleavage or trans-reactions as trueMichae-lis–Menten type enzymes.The biological reactions catalysed by ribozymes mainly

involve phosphodiester cleavage or transfer (i.e. religationto another nucleotide). The 2’ ribose hydroxyl group,characteristic of RNA, is always directly or indirectlyinvolved in catalysis. Acid–base catalysis has also been

O

O

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HO

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O N

OH

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OH

PO O–

HO

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O

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OH

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OH N+1

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SmallRibozymes

O

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–BH

OPOR

OS–

–OH

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H2C O

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N+1

OH

Introns

(b)

Group I Group II

O

HO

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OH+

+

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OP

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+

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(d)

Figure 1 The chemical pathway involves a site-specific attack of a nucleophile at the cleavage site. The nucleophile used differs with the ribozyme typeshowing the diversity of catalytic activity. (a) In the case of the RNAase P RNA the nucleophile is a free water hydroxide (OH2 ) or metal-boundhydroxide ion, which results directly in a site-specific hydrolysis with a 3’-OH and 5’-phosphate residue. (b) Small catalytic ribozymes are thought to usehydrated metals or possibly functional groups of RNA bases (e.g. ring nitrogens N1 of adenosine or the N3 of cytosine). The activation of the 2’-OHof the ribose 5’ to the scissile bond allows attack on the phosphate group leading to the formation of a cyclic 2’–3’ phosphate group and a newly formed 5’-OH group at the N11 base. Other small molecules, like imidazole, histidine, diamine, polyamines or even antibiotics, can probably substitute for thegeneral base catalyst. (c) Intron splicing of the group I ribozyme is initiated by the attack of the 3’-OH of the free guanosine cofactor (red). The ribose of thefree guanosine base becomes covalently linked to the 5’ end of the intron, and a new 3’-OH is formed at the 3’ end of exon 1. (d) In the case of group II intronthe 2’-OH group of the attacking adenosine residue is located within the RNA intron sequence itself (red). Upon self-splicing the attacking adenosine isadded covalently through a 2’–5’ linkage with the first base of the intron sequence.

Catalytic RNA

2 ENCYCLOPEDIA OF LIFE SCIENCES / & 2002 Macmillan Publishers Ltd, Nature Publishing Group / www.els.net

implied in the autocatalytic mechanism of the Hepatitisdelta virus (Nakano et al., 2000) as well as in the catalysis ofpeptide bond formation on the ribosome (Nissen et al.,2000).Ribozymes can be considered as metalloenzymes.

Indeed, the folding and/or catalytic activities of ribozymesoften depend on the presence of and interaction withdivalent metal ions (Pyle, 1993). Metal ions, depending ontheir atomic radius and ligand coordination geometry,bind to various sites in an RNA fold. The negativelycharged sugar–phosphate backbone generates a highdensity of negative potential in the deep groove. Inaddition, bases can interact directly with metal ionsthrough the exocyclic keto-substitutions and endocyclicring nitrogens or via water molecules to other functionalgroups. Magnesium ions are usual cofactors in catalyticRNAs, but it has been shown that some catalytic RNAsalso functionwith other divalent ions, likemanganese ions,in the absence of divalent ions or even in the presence ofhigh concentrations of monovalent ions. Polyamines arevery abundant in cells and are usually found to beassociated with nucleic acids. They can reduce therequirement for high cation concentrations and enhancethe cleavage rate or be catalytically active themselves.

Classes of Catalytic RNAs

tRNA processing by ribonuclease P RNA

The first example of an RNA molecule acting as a truerecycling catalyst is the ubiquitous endonuclease enzymeprocessing precursor tRNA 5’ ends, called RNAaseP (Figure 2a–c). Two structural classes of RNAaseP can be distinguished: type A is the ancestral subgroupfound in most bacteria, like Escherichia coli, and typeB is found in low (G1C)-containing Gram-positivebacteria (e.g. Bacillus subtilis). The E. coli RNAase Pconsists of a polypeptide (about 120 amino acids) anda 377-nucleotide RNA. Both of these are required forthe specific cleavage activity in vivo, but it is the RNAmolecule that catalyses the site-specific phosphodiestercleavage. The protein functions as an electrostaticshield, helping the RNA folding process into the activeconformation and promoting correct substrate bindingand positioning.Functional groups involved in catalysis are the 2’ ribose

hydroxyl group of the conserved pyrimidine (usually a U)at the position preceding the cleavage and a highlyconserved purine at the position of the cleavage on thepre-tRNA (Figure 1a). Also involved in the hydrolysisreaction are the nonbridging oxygens of the scissilephosphate. A nucleophile, a hydroxide or metal-boundhydroxide ion, attacks the phosphate immediately 5’ to themature tRNA. The hydrolysis reaction results in a 3’-

hydroxyl terminus and a 5’-phosphate terminus. Thecleavage reaction is dependent on divalent metal cations,like magnesium. Only in the presence of polyamines canmanganese, calcium or zinc ions substitute, albeit ineffi-ciently. It has been proposed that a hexacoordinatedmagnesium ion binds to the catalytic site on the RNAase PRNA; a hydroxyl ligand of the magnesium ion acts as ageneral base to catalyse hydrolysis of the substrate RNA.

Intron Splicing

All three major types of cellular RNAs, tRNA, rRNA andmRNA, are known to contain intervening sequences (IVS)or introns, which have to be precisely excised to producefunctional molecules or the right reading frame for proteinsynthesis. This process, known as RNA splicing, requiresthe recognition of the 5’ and 3’ exonic sequences, strandcleavage and ligation. Although most eukaryotic intronsare spliced by a complexmachinery, the spliceosome, someintrons in bacteriophages or organelles self-splice. De-pending on their secondary structure and cleavage they aredistributed in two main groups.

Group I introns

The most extensively studied ribozyme is derived from agroup I intron in the pre-rRNA gene of Tetrahymenathermophila. The nuclear pre-mRNA of the 23S rRNAgene can excise its 413-base intronic sequence in vitro in theabsence of proteins, but requires the presence of divalentcations and a guanosine cofactor.The guanosine cofactor provides the free 3’ hydroxyl

necessary to initiate a series of two transesterificationreactions resulting in intron splicing (Figure 1c). First, theexternal guanosine (or one of its 5’ phosphorylated forms)attacks the 5’ splice site, leaving the 5’ exon with a 3’-hydroxyl group. The second step is analogous to thereverse of the first step. The 5’ exon attacks the 3’ splice sitealways preceded by an invariant internal guanine residue.This results in ligation of the two exons. In both steps, thetransesterification reaction is associated with an inversionof the scissile phosphate configuration, indicating an SN2nucleophilic attack by the 3’-hydroxyl either of theguanosine cofactor or of the 5’ exon terminal residue(Figure 1c). The Tetrahymena group I ribozyme provides arate enhancement over the uncatalysed reaction ofapproximately 1011-fold, which is comparable to theenhancements seen with protein catalysts.The secondary structure (Figure 3a) and a three-dimen-

sional model of the catalytic core of group I introns(Figure 3b,c) have been proposed based on sequencecomparisons (Michel and Westhof, 1990). The model issupported by a wide range of experiments such as affinitycleavage by a guanosine substrate analogue, site-specific

Catalytic RNA


Catalytic RNA


photo-crosslinking, helix extension electron microscopyand low-resolution X-ray crystallography (Golden et al.,1998).The Tetrahymena ribozyme is a large molecule compris-

ing several structural domains (Figure 3a). It folds in thepresence of divalent cations, particularly magnesium ions.The domains can self-assemble to form the catalyticallyactive complex when synthesized separately. In vitromutagenesis and analysis of phylogenetic data show thatparts of the intronic sequence, the internal guide sequence(IGS), recognizes the 5’ and 3’ exons by base pairing(Figure 3a,b). The P4–P6 domain is an independentlyfolding unit. The X-ray crystallographic structure of thisdomain has been determined (Cate et al., 1996). Severaltertiary interactions between adjacent regions stabilize thecomplex. The folding of the ribozyme in the presence ofdivalent cations into an ‘active’ conformation is crucial forthe cleavage event.

Group II introns

In group II intron splicing, the excised intron is released asa branched lariat RNA involving an atypical 2’–5’phosphate linkage (Figure 1d). Group II introns have beenfound so far in the RNA of cell organelles in fungi,photosynthetic eukaryotes and bacterial genomes, wherethey interrupt genes of tRNAs, rRNAs and mRNAs. Thegroup II intron RNAs have been shown to self-splicein vitro in the absence of proteins but normally undernonphysiological salt and temperature conditions. In vivo

self-splicing depends on stabilization by helper proteins,but the ribozyme activity lies within the group II intronRNA itself. Group II introns are also shown to be mobileelements able to transpose into new insertion sites andhoming into intron-less alleles of the genome.Group II intron self-splicing proceeds by two consecu-

tive transesterification reactions similar to the reactions ofthe group I ribozymes. The major difference occurs in thefirst step, where the 2’-hydroxyl group of the attackingribose comes from an internal adenosine within the intronsequence (Figure 1d) as in nuclear mRNA splicingperformed by the spliceosome. Subsequently, the liberated3’-hydroxyl group of the 5’ exon attacks the phosphodi-ester at the junction between the intron and 3’ exon,resulting in a ligation of the two exons and the formation ofa lariat structure of the excised intron. Efficient intronsplicing depends on the stability of the base-pairingbetween tertiary elements. Further, it has been shown thatthe self-splicing requires a completion of the group IIintron before an efficient binding of the exon can beestablished, assuring the correct folding into the activetertiary conformation necessary for intron splicing.Two major subdivisions (IIA and IIB) have been

classified based on small sequence and structural differ-ences. Six subdomains, called D1–D6, can be determinedin the secondary structure but there is little sequenceconservation between different group II introns, except thehighly conserved attacking adenosine in D6. Conservedsequences are found in single-stranded regions around thecentral wheel close to the intron-binding site, domain D5and some sites in domain D1.

Figure 2 Note: the sequence of the RNA ribozyme is given only if the nucleotide involved is conserved (where A is adenine, G is guanine, C is cytosine, U isuracil, R is purine, Y is a pyrimidine, W is A or U and N stands for any nucleotide). Nucleotides that form standard Watson–Crick base pairs (blue) areindicated by little black lines, while non-Watson–Crick base pairs (red) are shown as little black circles. Regions forming a continuous helix of double-stranded and base paired nucleotides are shown on a blue background and labelled with P (or according to the most commonly used terms) and a numberaccording to its positionon theRNA strand counting 3’ to 5’. Regionswhich are formally single-stranded are highlighted bya red background, while regionsinvolved in a tertiary interaction are yellow. Single-stranded regions are usually labelled with J for junction and identified by the neighbouring helix regions,i.e. J15/16 is a single-stranded region between helices P15 and P16. Black arrows are used for better visualization of the connectivity of the RNA strandindicating the 3’ to 5’ direction. Dashed lines are used to highlight a tertiary interaction between two distinct regions in the secondary structure. Terminalhairpins, which mostly add terminal stability or vary in length and sequencedepending on the species, are outlined in black. The site of cleavage is indicatedby a red arrow.

(a) The secondary and tertiary structures of the RNAase P RNA as proposed on a series phylogenetic comparison and biochemical data (Massire et al.,1998). The inspection of the secondary structure shows an irregulardistribution of double-stranded (labelled with P for ‘base paired’ counting from the 3’ to5’ terminus) and formally single-stranded regions (usually called junctions J). The 3’-CCA sequence of the precursor transfer RNA (tRNA) base pairs with theinternal bulge of J15/16 of the catalytic domain (marked in green). Another contact is made by the single-stranded regions of J10/11 and J11/12 of theribozyme forming the T-loop recognition site of the pre-tRNA. The 5’-leader sequence of the pre-tRNA, which will be cut off, is in close proximity to thesingle-stranded region of J18/2.

(b) The view of the tertiary model of the RNAase P RNA together with its pre-tRNA (shown in white) shows the structural organization after correct foldingof the ribozyme RNA. The ribozyme is composed of two different structural domains, the T-loop recognition site located in the upper half of the moleculeand the catalytic domain including the acceptor stem recognition site and catalytic core in the lower half. Both domains are held together by several tertiarycontacts (yellow). All functional single-stranded regions are now in close proximity to each other and outline the inner cave created by the ribozyme(marked in red) facing towards the pre-tRNA. Elements responsible for tertiary contacts, which hold the two subdomains together, are situated on theoutside of the ribozyme (yellow). Double-stranded regions (blue) are located mainly within the molecule providing the framework for correct folding andpositioning of functional groups and tertiary contacts.

(c) The inside of the RNAase P facing towards the tRNA (tRNA not shown) is mostly lined out with single-stranded regions responsible for recognition,correct alignment and catalytic activity. The two subdomains of the molecule are held together by several tertiary contacts, which are situated mainly onthe backside of the molecule. The inset depicts the position of the tRNA (shown in white) sitting on the RNAase P RNA molecule.

Catalytic RNA


Figure 3 See note to Figure 2. (a) The secondary structure of the TtLSU group I intron of the pre-rRNA of Tetrahymena thermophila, just prior to the secondstep, i.e. exon ligation, shows the modular composition. The internal guide sequence (IGS, P1 and 10) is base pairing with the 5’ and 3’ exon(lower case) shown in green. The cleavage site is specified by an arrow (magenta). (b) The stereoview of the complete tertiary model shows thecompactness and tight packing made possible by several tertiary interactions on the outside of the molecule (Lehnert et al., 1996). The P4–P6 region formsan independent subdomain, which can self-assemble and provide stability for the catalytic core. Several tertiary interactions with adjacent regions stabilizethe complex from the outside by bulge–bulge base pairing, like the P13 and P14 located along the equator, or the GNRA apical loop/receptorinteraction, as the P5/L9bcontact visible at the top. (c) This view shows the core of the group I intron from the reverse site. The IGS (green) is sitting on top ofthe helical stacks formedby the P4–P6 and P7–P3–P8 subdomains. The catalytic pocket is surrounded by nucleotides A261 and A314, and bases involved ininternal tertiary interactions form the base of the active centre, like G108, C109, G212, and U259. The platform of the cofactor guanosine-binding site(orange) is in close proximity to the cleavage site (magenta).

Catalytic RNA


Small Catalytic RNAs

Amongst the simplest catalytic RNA molecules are thesmall nucleolytic RNA species. Each undergoes self-cleavage and has a well-defined tertiary structure in thepresence of divalent cations. The hepatitis delta virusribozyme is a small RNA pseudoknot, which undergoesself-cleavage in vitro in the presence of magnesium ions,and is found in the genomic and antigenomic RNAs of thishuman pathogen. The Neurospora VS RNA is derivedfrom the mitochondria of Neurospora isolates and hasseveral helical elements, which fold in the presence ofmagnesium ions. The self-cleavage event occurs in a stem–loop region, within a small symmetric loop. A number ofsmall plant pathogenic viroids and virusoids have beenisolated fromplants. These ribozymes undergo site-specificself-cleavage in the presence of magnesium ions, which isrequired for the generation of the unit-sized RNAmolecules following rolling circle replication.Among theseare the tobacco ringspot virus RNA and its associatedsatellite RNAs. The ‘hammerhead’ motif was identified onthe positive (1) strand of the satellite RNA of the tobaccoringspot virus RNA, while on the negative strand adifferent structural motif called ‘hairpin’ was discovered.

The hammerhead ribozyme

The hammerhead ribozyme is the smallest known ribo-zyme. All conserved bases are in the single-strandedregions linking the helical stems and terminal loops addstability and ensure correct folding (Figure 4a). The three-dimensional structure obtained by X-ray diffractionstudies (Pley et al., 1994) shows thatmost of the nucleotidesin the single-stranded regions base-pair to formaY-shapedstructure.The folding into the active tertiary structure (Figure 4b)

proceeds in a two-step transition by the consecutivebinding of two single cations. The hammerhead ribozymealso requires divalent cations for self-cleavage, withmagnesium and manganese ions being the most efficient.The self-cleavage reaction occurs by a transesterificationreaction using an in-line SN2mechanism, where the scissilephosphorus bond is attacked by the 2’-hydroxyl groupbelonging to the 5’ ribose (Figure1b). The cleavage productsare 2’-3’-cyclic phosphate and 5’-hydroxyl termini. A singlesubstitution of deoxyribose sugar for the ribose at thecleavage site abolishes cleavage. Chemical substitution hasbeen used to probe the role of metal ion cofactors incatalysis. The results indicate that one metal ion is boundto the pro-R oxygen atom of the phosphate. The transitionstate of the SN2 reaction requires a colinear alignment ofthe phosphorus atom, the attacking and the leaving oxygenatoms and, thus, part of the mechanism of rate enhance-ment could be a distortion of the local RNA structure tofacilitate the achievement of this alignment (Figure 1b).

The hairpin ribozyme

The hairpin ribozymemotif is the second smallest catalyticRNA molecule with a functional length of 50 nucleotides.It catalyses not only a cleavage reaction but also its reversereaction (ligation). The catalytic motif consists of twocatalytic internal loops, loop A and B. These loop regionsare connected in its natural form by a four-way RNA

Figure 4 See note to Figure 2. (a) The secondary structure of thehammerhead ribozyme illustrates the folding of the two domains. Thehammerhead ribozyme is the smallest and simplest of the small catalyticRNAs. Non-Watson–Crick interactions (red), together with structural metalions, stabilize the native fold. A two-step transition of the molecule isobserved by the consecutive binding of two single cations to the twodomains within the ribozyme. Conserved bases are specified. The cleavagesite is shown in magenta. A terminal hairpin loop, which is not necessary forcleavage activity, is outlined in black. (b) The X-ray structure (Scott et al.,1995) shows that domain II forms upon the continuous stacking of stem IIIon stem II. Structural metal ions bind to A9, A13 or A14 and stabilize atandem of G–A base pairs, widening the deep groove. The formation of theactive hammerhead ribozyme does not involve any tertiary interaction incontrast to all other known natural ribozymes.

Catalytic RNA


junction, which can be reduced to a hinge in a minimizedform still able to cleave in trans (Figure 5a).Most of the bases and functional groups essential for

catalytic activity are found within the two loops. Nuclearmagnetic resonance (NMR) structures of the two domains

have been solved separately showing extensive base pairingwithin the two internal loops. A three-dimensional modelof the hairpin ribozyme has been proposed based on therelative distances obtained from the crosslinking experi-ments (Figure 5b) (Earnshaw et al., 1997). Fluorescenceresonance energy transfer (FRET) experiments suppliedthe first physical evidence for a loop–loop contact betweenthe catalytic domains A and B for the hinged construct(Walter et al., 1998) and in its natural context as a four-wayjunction. Transient electrical dichroism measurementsrevealed a bending angle of approximately 808+208between the two domains upon tertiary interaction.The metal ions involved in the folding of the hairpin

ribozyme are probably required for structural purposesonly, aligning the catalytic domains and defining a highlyspecific architecture of the catalytic pocket. Perhapsfunctional groups of the RNA participate directly in thecatalytic chemistry andmetal cofactors play amore passiverole, as proposed for the hepatitis delta virus ribozyme.

Human Hepatitis delta virus

The human pathogen Hepatitis delta virus (HDV) is asmall, single-stranded satellite virus of hepatitis B(Figure 6a,b). The single-stranded RNA genome is about1700 nucleotides long. It is associatedwith a high incidenceof fulminant hepatitis and death in infected humans. Likecertain pathogenic subviral RNAs that infect plants, HDVRNA features a closed-circular conformation, a rolling-circle mechanism of replication and RNA-catalysed self-cleaving reactions of both genomic and antigenomicstrands in vitro important for viral replication.The minimal natural ribozyme is about 85 nucleotides

long. It adopts a secondary structure with four pairedregions (PI–PIV) forming an atypical pseudoknot second-ary-structure arrangement (Figure 6a). Computer-assistedmodelling provided the first three-dimensional structure inwhich residues C75, U20 and C21 form the basis of thecatalytic region and are close to the cleavable phosphate.The crystal structure of the HDV (Figure 6b) shows that anadditional pseudoknot is formed through the formation ofanother 2-bp helix (PI.I) involving residues of loop L3(Ferre-D’Amare et al., 1998).There is probably no direct role for a metal ion in

catalysis. The cytosine at position 75 in the genomicsequence (C76 in the antigenomic) provides an aminogroup, which can accept a proton from the 2’-hydroxylgroup of the nucleotide 5’ from the cleavage site (Figure 1b).The RNA itself acts therefore as a general acid–basecatalyst (Nakano et al., 2000).

Neurospora VS ribozyme

The Neurospora varkud satellite (VS) ribozyme found inthe mitochondria of the Varkud-1c natural isolate of

Figure 5 See note to Figure 2. (a) The secondary structure of the hairpinribozyme stresses the almost parallel alignment of the two arms. In itsnatural context it has a four-way junction at the centre of the hinge.Conserved bases and important functional groups are mainly located intothe formally single-stranded loop regions (red). The cleavage site isindicated by a magenta arrow. The hairpin loop which adds terminalstability only is outlined in black. (b) Molecular modelling proposes that thetertiary interaction of the two loops is established by a ribose zipper motif(yellow) (Earnshawet al., 1997). It is probably this tertiary interactionwhichforms the catalytic pocket. G36–U37–A38 is now in close proximity to thecleavage site (magenta). Perhaps functional groups within RNA participatedirectly in the catalytic activity, since an inner sphere complexof a metal ionis not required.

Catalytic RNA


Neurospora differs in both sequence and secondarystructure requirements from other ribozymes mentionedabove. It is one of the largest ribozymes, comprising acatalytic core within a region of 153 nucleotides down-stream and upstream from the cleavage site. Up to datethere is only a limited amount of structural informationavailable. The proposed tertiary structure is thought toaccommodate a long-range pseudoknot formed by loops Iand V (Rastogi et al., 1996). The VS RNA also has theability to recognize a stem–loop substrate RNA in a trans-cleavage reaction.

Artificial Ribozymes

Natural catalytic RNAs are limited to reactions involvingphosphoryl centres.Novelmechanisms are currently underinvestigation. As a result a huge variety of artificialribozymes have been selected by in vitro selection. Thishas yielded ribozymes using different catalytic metal ioncofactors. In addition to cleavage reactions the catalysedreactions include phosphorylation, ligation, polymeriza-tion and transesterification. They attack phosphoryl,carbonyl or alkyl centres.Ribozymeswith ribosyl-cleavageproperties, a ligase activity (3’–5’, 2’–5’ and 5’–5’ phos-phodiester linkage) and with a phosphorylation reactioncould be selected. Other artificial ribozymes include self-aminoacylation, acyl transfer and aminoacyl transferreactions, self-nitrogen and sulfur alkylation, biphenylisomerization or porphyrin metalation. Even a ribozymecatalysing a carbon–carbon bond formation by the Diels-Alder reaction could be selected.

Interaction with Antibiotics

A variety of antibiotics have been found to interact withRNA molecules. Antibiotics inhibit RNA function intranslation as in the case of 16S rRNA being part of theribosome apparatus or viral RNA replication by blockingthe Rev-RRE or Tat-TAR interaction of the human HIVvirus. Aminoglycoside antibiotics are shown to bind tocatalytic RNAs and inhibit their activity. In two cases, thehairpin ribozyme and the Neurospora VS ribozyme,aminoglycosides can stimulate cleavage activity in theabsence of metal ions.The inhibition depends on the number and distribution

of positively charged amino groups displacing importantstructural and/or catalytic metal ions from the RNA. It isthought that the aminoglycosides are in competition withother cofactors or prevent the correct folding into thecatalytic active architecture or intervene with the RNA–substrate complex formation. It has been shown that geneexpression of an mRNA can be controlled in living cellsafter insertion of a small RNA aptamer sequence into the

Figure 6 See note to Figure 2. (a) The secondary structure of the genomicstrand of the HDV ribozyme is shown here. The ribozyme is composed offour paired regions called PI–PIV (blue) with PII and the additional PI.Iforming two atypical pseudoknot structures (yellow). Conserved bases arespecified. The cleavage site and the last base of the 5’ end are indicated inmagenta. The hairpin stem–loop PIV (black) functions as a clamp, addingterminal stability and can be exchanged. (b) The X-ray structure (Ferre-D’Amare et al., 1998) shows that the molecule adopts a compactconformation, where helix PI stacks upon PI.I and PIV, which is in a side-by-side orientation to the colinear stacked helices PII and PIII. Thisarrangement places the active site right in the centre of the catalyticallyimportant bases within the single-stranded regions (red). The tertiaryinteractions of the two pseudoknots form the catalytic pocket. U20, C21and C75 are in close proximity to the cleavage site (magenta).

Catalytic RNA


expression regulation region by the interaction with aligand molecule, like an antibiotic. This could in principlebe used for the timed and targeted expression oftherapeutic ribozymes.

Therapeutic Use of Ribozymes

Extensive research has attempted to exploit the propertiesof RNA cleavage for practical use (Usman et al., 1996).Ideal candidates are the small catalytic RNAs because oftheir size and the low number of conserved nucleotidesneeded on the ribozyme strand, while at the same time therest of the substrate sequence can be used for increasedspecificity. Also they can be chemically synthesized easilyand have natural activity at cellular ionic conditions.Finally, the drug delivery and cellular uptake have to bedetermined and a high turnover rate maintained forefficient catalysis. The key to the future availability ofthese products is the chemical stabilization and protectionof these RNAs against natural degradation in the cell.The ribozymes most suitable for cleavage of RNAs in

trans are the hammerhead ribozyme and the hairpinribozyme. Of major attraction for the application ofribozymes in gene therapy is the possibility to specificallycleave RNA of human viruses, such as the genomic RNAof the HIV virus causing AIDS in humans (Macphersonet al., 1999), the human pathogenic hepatitis B and Cviruses (Welch et al., 1996), themumps virus (AlbuquerqueSilva et al., 1996) or the influenza A virus (Tang et al.,1994). Carcinogenic mRNAs, like the c-Ha-ras mRNA(Koizumi et al., 1993) or the bcr-abl oncogene mRNA(Snyder et al., 1997) responsible for two kinds of leukaemiahave also been targeted (Verfaillie et al., 1999). Otherexamples of the use of ribozymes include the degradationof Alzheimer-causing amyloid peptide pre-RNA (Denmanet al., 1994), enhancement of T-cell response (Cepero et al.,1998) by inhibition of a negative regulatory receptor, therescue of photoreceptor cells (retinitis pigmentosa blindingdisease) (Lewin et al., 1998) and the inhibition of cellgrowth of human cervical keratinocytes (Alvarez Salaset al., 1998). TheHDV ribozyme is also known to cleave anRNA substrate in trans. Even a variety of catalytic DNAoligonucleotides have been obtained, which cleave chi-maeric oligonucleotides at a single ribonucleotide positionembedded within a deoxyribonucleotide context.Theuse of ribozymes against naturalRNAtargets in vivo

depends on a variety of parameters. First, the transportinto the human body has to be established. Severalstrategies have been employed: (1) in vivo exogenousdelivery of ribozymes, in which ribozymes are mixed withcationic lipids and then injected into the peritoneal cavity;(2) in vivo injection of synthetic ribozymes directly, e.g. intothe synovium of a knee to act against active jointdegradation by osteoarthritis; (3) ex vivo gene transfer bytransfection, where a ribozyme gene construct is delivered

to the cell by transfection of a cell line by modifiedretroviruses and the infected cells are retransferred into thehuman body; and (4) ex vivo gene transfer by transductionof ribozyme genes in which ribozymes are transduced intolymphocytes or progenitor cells, which give rise tolymphocytes or other cell lineages, and reintroduced intothe human body, targeted against HIV infection. The nextstep is cellular uptake through the cell membrane.Once in the cell, the target RNA has to be recognized

before the ribozyme associates with its substrate. Theassociation has to be specific for the targeted RNA onlyand the complex has to be stable. This leads to a temporaryinactivation of the target RNA by functioning likeantisense RNA. In addition, ribozymes are able to cutthe target RNA into two nonfunctional RNA products,which are then degraded by the cell. The chemical cleavagereaction of the ribozyme needs to be optimized to functionunder cellular conditions, including salt concentration,pH, temperature and conformational changes of thetertiary structure of theRNA.After the substrate cleavage,the two product strands have to be quickly released anddissociated to guarantee rapid turnover. The intracellularexpression, stability and kinetic activity of ribozymes havetherefore to be optimized.With the hammerhead ribozyme, the consensus se-

quence for the cleavage site in the target RNA has tocontain a 5’GUC*N 3’motif and for the hairpin ribozymea 5’ YN*GUC 3’ motif is necessary, where Y represents apyrimidine base, N can be any base and the asteriskindicates the site of cleavage. The cleavage site has to besituated in an accessible region within the tertiary fold ofthe targetRNApreferably in a hairpin loop. The sequencesflanking the cleavage site are not important as long asstable base pairs are maintained, but helix lengths are veryimportant, since they control sequence specificity, rate ofassociation and dissociation of products.The stability of the ribozyme RNA against ribonuclease

attack can be increased by several chemical modificationsof the ribose sugar (addition of a 2’-methyl, 2’-amino, 2’-C-allyl group or a 3’-3’ linked deoxythymidine). Anotherconcept is the use of modified purine bases or pyrimidinebases. Further, the phosphates can be modified byreplacementwith phosphorothioates or phosphoamidates.In vitro selection methods can be used to isolate ribozymeswith increased activity against substrates. Some ribozymeshave been shown to have undesired cytotoxic side effects.The problem of escape mutants due to the high mutabilityof viruses can be overcome by the introduction of amultitargeting strategy cleaving the target RNA at multi-ple conserved sites.

Final Remarks

Catalytic RNAs are widely found in nature and performimportant reactions within the cell. These tasks include the

Catalytic RNA


correct processing of functional messenger RNAs byintron excision, the translation step during the maturationof transfer RNAs by the ribonuclease P RNA and the self-replication and multiplication of viral RNA genomes,including human and plant pathogens, during their lifecycle. Recently, it has been shown that protein synthesis iscatalysed within the ribosome at the level of a ribosomalRNA. In addition, the selection of artificial ribozymes hasexpanded the use of catalytic activity beyond their naturalcounterparts. The catalytic action of ribozymes can becontrolled by antibiotics, an observation that provides anew strategy for antiviral and antibiotic treatment in thehealth sector. The therapeutic use of ribozymes for humangene therapy will be of major importance, extendingexisting treatments by vaccines and antibiotics. Ribozymescan provide a highly specific agent against viral RNAs orcarcinogenic and unwanted gene products.

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Further Reading

EcksteinFandLilleyDMJ (eds) (1996)CatalyticRNA. In:NucleicAcids

and Molecular Biology, vol. 10. Berlin: Springer-Verlag.

Ferre-D’Amare AR and Doudna JA (1999) RNA folds: insights from

recent crystal structures. Annual Review of Biophysics and Biomole-

cular Structure 28: 57–73.

Gesteland RF and Atkins JF (eds) (1993) The RNA world. In: Cold

Spring Harbor Monograph Series, vol. 24. New York: Cold Spring

Harbor Press.

Catalytic RNA


Green R and Schroeder R (eds) (1996) Ribosomal RNA and group I

introns. In: Molecular Biology Intelligence Unit, vol. 38. New York:

R.G. Landes and Chapman & Hall.

GoodPD,KrikosAJ,Li SX et al. (1997)Expression of small, therapeutic

RNAs in human cell nuclei. Gene Therapy 4(1): 45–54.

Jaeger L (1997) The new world of ribozymes. Current Opinion in

Structural Biology 7: 324–335.

Lilley DM (1999) Structure, folding and catalysis of the small

nucleolytic ribozymes. Current Opinion in Structural Biology 9(3):

330–338.

Michel F andFerat JL (1995) Structure and activities of group II introns.

Annual Review of Biochemistry 64: 435–461.

Morgan RA and Anderson WF (1993) Human gene therapy. Annual

Review of Biochemistry 62: 191–217.

Tsang J and Joyse G (1996) In vitro evolution of randomized ribozymes.

Methods in Enzymology 267: 410–426.

Tanner NK (1999) Ribozymes: the characteristics and properties of

catalytic RNAs. FEMS Microbiology Review 23: 257–275.

Walter F, Vicens Q and Westhof E (1999) Aminoglycoside-RNA

interactions. Current Opinion in Chemical Biology 3: 694–704.

Catalytic RNA


catalytic rna · otherrnaoligonucleotidesformingpseudoknots,kissing loopsormultistrandedhelices....

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