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Twenty years ago, it became clear that ribonucleic acids,or RNAs, are used as catalysts in living cells, in additionto their known roles in information storage and as molec-ular architectural frameworks. This idea was soprofoundly contrary to the central dogma of molecularbiology that it resulted in the award of a Nobel prize totwo of the early proponents, Thomas Cech and SidneyAltman. It has inspired what is now itself the dogmaticview of pre-biotic macromolecules, the ‘RNA World’hypothesis (Gesteland et al., 1999). This hypothesisstates that RNA, or something like it, was the originalmacromolecule, capable of both encoding its own repro-ductive information and catalysing the essential chemicalreactions that effect reproduction. The RNA World is acompelling vision in that it offers a solution to the‘chicken-and-egg’ problem of how to simultaneously bringinto existence the complex processes of encoding geneticinformation in nucleic acids while decoding them intotheir ‘functional’ products, the enzymes. If a singlemacromolecule could be both the genetic carrier and thecatalyst, decoding into the more modern catalysts,proteins, could have evolved later.

While proteins have largely taken over as the biologi-cal catalysts of the modern world, there are stillremnants of the RNA World to be found. Cech, Altman,and others originally characterised them in isolated RNAprocessing reactions, but recent work suggests that RNA

catalysts are still at the heart of modern RNA andprotein synthesis.

Ribozymes that cut themselves or other RNAsThe original discovery of ribozymes by Cech and Altman wastwofold – RNA segments that cut themselves out of largerRNAs (self-splicing introns) and a protein-assisted RNAenzyme (ribonuclease P) that cuts the leader sequences off alltransfer RNAs throughout the three organismal domains.(The first two are the Nobel lectures: Altman, 1990; Cech,1990; Gesteland et al., 1999). This instigated a hunt forcatalytic RNAs with a function in the biological processing ofRNA. Quickly, a large number of self-splicing or self-cleavingRNAs were identified that make single cuts in preciselydefined RNA sequences (the difference being that self-splic-ing RNAs go further and rejoin the ends of two single cuts)(Table 1). Although the reactions are often facilitated byprotein cofactors in vivo, the active enzyme in each case is theRNA component. Two general mechanisms are recognised forsplicing and cleavage reactions, characterised by the kinds ofintermediates and products they generate. These mecha-nisms, shown in Figure 1A and B, differ primarily in that the‘A’ mechanism uses an external hydroxyl group (from wateror a nucleoside) as the backbone cutting nucleophile, whereasthe ‘B’ reaction uses the 2’-hydroxyl from the sugar immedi-ately preceding the scissile phosphodiester.

Ribozymes:Catalytic RNAs that cut things, makethings, and do odd and useful jobs

Nils G Walter and David R EngelkeThe University of Michigan, Ann Arbor, USA

Catalytic RNAs, or ribozymes, are a fossil record of the ancient molecular evolution of life onEarth and still provide the essential core of macromolecule synthesis in all life forms today.Are they also an avenue to the development of new catalysts to recreate evolution, or to use

as therapeutics and molecule sensors?

Ribozymes:Catalytic RNAs that cut things, makethings, and do odd and useful jobs

200 Biologist (2002) 49 (5)

In a catalytic RNA up to hundreds of nucleotides inlength, only a single bond is cut. How can an RNA catalystachieve this level of precision? Soon RNA biochemistsstarted to employ tools extensively developed for proteinenzymology to explore this conundrum. One of the first‘tricks’ they taught their new pets was to cut an externalsubstrate in ‘trans’, rather than themselves in ‘cis’. Withthis advent, these ribozymes became true catalysts, facili-tating repeated substrate reactions without being modifiedthemselves (Figure 2). Besides providing a powerfulavenue to ribozyme-mediated gene inactivation (seebelow), trans-acting ribozymes are amenable to the

complete arsenal of enzymology, including pre-steady-state and steady-state kinetic analysis.

Quickly, ‘ribozymology’ focused our attention on one ofthe most dominant properties of RNA as a biopolymer, itshigh negative charge (one charge per nucleotide). As aconsequence, the presence of positively charged cations isessential for the catalytic activity of all ribozymes. In thecell, the cation of choice is Mg2+, as it has a high affinity forthe negatively charged phosphate backbone of RNA and isthe most abundant divalent metal ion (one to two mM freeconcentration). Its double charge enables bridging of twophosphates from distant RNA regions, facilitating long-

range structure formation. Indeed,RNA catalysts can form intricatethree-dimensional structures thatrival in complexity those of proteinenzymes, allowing for the precisepositioning of a substrate in acatalytic core (Figure 3). In addi-tion, Mg2+ in aqueous solution canform the deprotonated Mg(OH)(aq)+

complex that, due to its residualpositive charge, still binds to RNA.This relieved the chemists’ concernthat, at first sight, the four nucle-obases of RNA (guanine, adenine,cytosine, uracil) seemed quitelimited in their ability to do all thechemistry outlined in Figure 1. Aproperly positioned Mg(OH)(aq)+

ion, acting as a base catalyst, couldeasily be envisioned as an essentialcofactor for most of these reactions.Much to the dismay of RNAbiochemists, ribozymes were thusquickly dismissed as chemicallyinferior to proteins, which sport 20versatile aminoacid side chains.

However, in recent years, severalribozymes have taught us newlessons about their catalytic

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Table 1. Natural ribozymes and what they do

Ribozyme Number identified Biological source Reaction catalysed (product)

Group I introns >1000 Eukaryotes (nucleus and mitochondria), Self-splicing transesterification (3’-OH)prokaryotes, bacteriophages

Group II introns >700 Eukaryotes (organelles), prokaryotes Self-splicing transesterification (3’-OH)

Group-I intron like 6 Didymium, Naeglaria Hydrolysis (3’-OH)

RNase P RNA >300 Eukaryotes (nucleus and organelles), Hydrolysis (3’-OH)prokaryotes

Hammerhead ribozyme 11 Plant viroids and satellite RNAs, newt Self-cleaving transesterification( 2’,3’-cyclic phosphate)

Hairpin ribozyme 4 Plant viroids and satellite RNAs Self-cleaving transesterification(2’,3’-cyclic phosphate)

Hepatitis delta virus 2 Human hepatitis delta virus Self-cleaving transesterificationribozyme (2’,3’-cyclic phosphate)

VS ribozyme 1 Neurspora mitochondria Self-cleaving transesterification( 2’,3’-cyclic phosphate)

Ribosomal RNAs >5000 Eukaryotes, prokaryotes Peptidyl transfer (peptide bond)

Spliceosomal RNAs >100 Eukaryotes Trans-splicing transesterification (3’-OH)

R'

O

H

OBase -1

(O)HO

P

O

O-O

3' R

5' R

OAdenosine

OHO

5' R

C O

CH

NH

R'

R

OBase -1

OHO

P

O

O-O

3' R

5' R

N

O

O

R'

R

:B

R'

O

H

OBase

OHO

3'R

O

P

O

O-O

P

O P O-

O

-O

-O

O

Nu:

E

A B C

D F

O

O

O

O

Figure 1. The catalytic portfolio of ribozymes. (A) Hydrolysis (R’ = H) or transesterification (R’= organic residue) of an RNA or DNA phosphoester linkage. R’ = H for RNase P, R’ = guano-sine for group I introns, R’ = internal adenosine for group II introns. (B) RNA cleavage ascatalysed by the small hammerhead, hairpin, and HDV ribozymes (B=base). (C) RNA chainelongation. (D) Peptidyl transfer (Nu = NH2 group of another amino acid), amide bond forma-tion (Nu = 5’-NH2 of a modified RNA), or ester hydrolysis (Nu = water). (E) Isomerisation. (F)Diels-Alder cycloaddition.

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prowess. One such lesson is that larger ribozymes can notonly precisely position an RNA substrate in a pre-organ-ised active site, but also similarly position a secondsubstrate such as a nucleoside to react it with as a nucle-ophile. Hence, RNA can drive catalysis by forced proximityof reaction partners (Gesteland et al., 1999).

A second and completely unexpected lesson of recentyears is the fact that ribozymes have found ways to utilisetheir own side chains directly to do chemistry (Butcher,2001). Two examples from small catalytic RNAs, the hair-pin and hepatitis delta virus (HDV) ribozymes, are shownin Figures 2 and 3. In both cases, the ribozyme preciselyfixes the location of its substrate by hydrogen bonding in aspecific binding pocket. It then employs one of its nucle-obases, whose protonation equilibrium it fine-tunes formaximum reactivity around physiologic pH, to exchange aparticular proton with the substrate, inducing a specificcut. The human analogy that comes to mind is that of anable blacksmith, holding his metal to an anvil to ply it withhis hammer in a particular spot. No wonder that catalyticRNA is so precise and adept.

One particularly interesting aspect of these smallribozymes that are engineered to cut in trans is theirstraightforward targeting mechanism – Watson-Crickbase-pairing between sequences flanking the catalyticdomain and sequences surrounding the cleavage site. Thismeans that the target RNA substrate can be varied bysimply changing these targeting sequences (Figure 2). Thisprovides the ability to easily create enzymes, ‘designerribozymes’, to repeatedly cleave and inactivate specificRNA sequences. This idea has particularly caught theattention of researchers hoping to inhibit virus infection oroncogene function by providing these tailored ribozymes tohuman cells (Lewin & Hauswirth, 2001).

Pervasive use of RNA in synthesis ofmacromolecules The use of RNA in protein synthesis has long been part ofthe central dogma. Not only is information carried inmessenger RNA (mRNA) triplet codons, but transfer

RNA (tRNA) serves as the adapterto interpret codons into aminoacids; and the enormously complexribosome, containing both riboso-mal RNA (rRNA) and proteincomponents, serves as the factoryfor decoding the message intoprotein chains. In addition, the last20 years have seen a steady rise inthe number of distinct small RNAsthat are known to be used in macro-molecular synthesis and regulation.Small nuclear RNAs (snRNAs) andsmall nucleolar RNAs (snoRNAs)are essential subunits of a largeand diverse group of RNA-proteincomplexes (RNPs) that process pre-mRNAs and pre-rRNAs, respec-tively. Small cytoplasmic RNAs(scRNAs) include the 7SL subunitof the signal recognition particle,an RNP involved in nascent proteintranslocation from ribosomes intothe endoplasmic reticulum.Finally, complexes betweenproteins and small double-strandedRNAs have recently been discov-

ered as the basis of powerful and universal pathways ofcellular gene regulation and antiviral defense, calledRNA interference (RNAi).

Although a high percentage of RNA processing reac-tions appear to involve RNPs, it is unclear exactly whatcatalytic elements may reside in the essential RNA

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AA

UAU

N

G A

N

A

NCN

N

N

3'

Helix 3

Helix 4

Hinge

Loop BHelix 2

N

N N

AG

AYG

Y R

3'5'

Helix 1

Loop A G

A

U

5’

Hairpinribozyme

Stem I

Stem II

A

AG

3' 5'

Stem III

G

A

A

3'

5'U

UU

C

CA

G

Hammerheadribozyme

AU

N

G

A

C

3'

P2

P4

P3G

G

P1

G

U

C

CU

G

C

CC

3'

G

5’

5’

HDVribozyme

P1.1

LegendEssential Guanosine

Essential Cytosine

Essential Adenosine

Essential Uridine

Two H-bond interaction

Essential G or A

Standard Watson-Crick base pair

Single H-bond interaction

A

G

U

C

N

R

Y Essential C or U

Any nucleotide

Figure 2. What they look like: Schematic representations of the three-dimensional structures ofthree small ribozymes, engineered to act in trans on external substrates of choice. Long dashedlines, sequences removed to generate the trans-acting ribozymes; gold, substrates; arrows, cutsites. Additional colours used in the hairpin and HDV ribozymes correlate with those used inFigure 3.

Hairpin ribozymegold: substrateblue: anvilred: hammerpurple: H-bonds

HDV ribozymegold: substrateblue: anvilred: hammerpurple: H-bonds

Figure 3. Caught in the act: Details of the catalytic core of the hair-pin and HDV ribozymes. The colour scheme is the same as inFigure 2. The red nucleotides are poised to act on the substrate byabstracting or donating a proton. Dashed purple tubes, hydrogenbonds to fix the substrate in place.

202 Biologist (2002) 49 (5)

subunits of these RNPs. In the case of mRNA splicing(intron removal), the RNP involved, called the spliceo-some, clearly reconstitutes the same reaction found inthe simpler, self-splicing ribozymes of the group IIintrons (Table 1). In fact, recent evidence suggests thatthe snRNA components are ribozymes themselves andcarry the catalytic activity of the spliceosome. Thus, anexternal RNA-containing complex has evolved to splice intrans, rather than each intron needing to carry its ownribozyme. Many of the modern functions of RNA mightsimply involve such cases, in which a function that origi-nally was performed by an RNA on itself has becomeexternalised.

For a while, interest in ribozymes had been limitedsomewhat due to the feeling that they were essentially a‘one-trick’ class of enzymes – they simply cut otherRNAs. While this left the aficionados with a lot of inter-esting RNA processing reactions, the RNA World hypoth-esis was waning in the general scientific community. Forone thing, RNA would have had to accomplish a greatmany things, especially the synthesis of its eventualsuccessors in molecular evolution, proteins. In thiscontext, there was much rejoicing when a series of papersover the last few years established that the ribosome,that gigantic particle that was always thought to containa large RNA framework for structural reasons only, isreally a large ribozyme that catalyses peptide bondformation. While the modern ribosome has becomedependent on proteins for efficient function, it is clearthat the catalytic centre is still composed of RNA. Earlywork suggested that the deproteinised rRNA componentcould carry out the peptidyl synthetase reaction, andrecently published crystal structures show that theregion of the active center is solely composed of RNA(Figure 4), with protein components residing primarilyon the periphery of the complex (Cech, 2000; Nissen etal., 2000). As with the small hairpin and HDV ribozymes(Figure 3), a particular RNA nucleotide has been

proposed to exchange a proton with the substrate (Figure4, bottom), although this is still hotly debated within thefield (Moore & Steitz, 2002). Be that as it may, at itscore, protein synthesis is composed of two tRNA cofactorsbringing together the growing peptide chain and the nextamino acid in an RNA active site.

New RNA catalysts through forced molecularevolutionCan RNAs be used to catalyse additional reactions? Theanswer is emphatically yes, and new ribozymes arecurrently being identified through a process that is vari-ously called in vitro evolution or SELEX (SystematicEvolution of Ligands by EXponential enrichment (Wilson &Szostak, 1999)) (Figure 5). The idea is a simple one, givencurrent technology, and mimics natural evolution. A largepool (~1015–1017) of RNA or DNA molecules is synthesised, inwhich the sequences at the ends are known, but the centreis randomised. In this huge sequence pool, there is some-thing (usually a lot of things) that will bind to practicallyany molecular surface or fulfil any number of enzymatictasks to at least some extent. In its simplest form, RNA orDNA that binds to a molecular target is separated by one ofmany methods from the great majority of molecules that donot bind. Because the end sequences of all molecules are thesame and known, it is possible to amplify the binders by avariation of PCR (polymerase chain reaction) called RT-PCR(reverse transcription followed by PCR) to get sufficientphysical quantities of the binding molecules to characterise.Typically, the winners of the first round are sent through atleast several rounds of re-selection, often with deliberatemutagenesis, to obtain winners with optimised bindingcharacteristics, so-called aptamers.

The main limitation when applying this technique toidentify new ribozymes is the ability to sort the smallnumber of molecules that perform a particular functionfrom the large number that do not. This is relativelystraightforward when the desired function is tight bindingto a molecule that can be immobilised on a bead or surface.However, when searching for an enzyme, it is necessary to

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Figure 4. A ribosome’s true colours. (Top) The large subunit of theribosome, with proteins in blue and RNA components in orange,grey, and burgundy. Green and red ribbons, tRNAs. (Bottom) Thepeptidyl transfer mechanism catalysed by the RNA components ofthe ribosome. A particular adenosine (A2451 in Escherichia coli) isrendered unusually basic by its environment within the folded struc-ture; it is presumed to act as a base and abstract a proton as shown.Reprinted with permission from T R Cech (2000). Copyright 2000,American Association for the Advancement of Science.

START:

Library1017 different RNA

molecules

Select for ability to perform a

function (e.g., catalysis, binding)

Winners(<102)

Losers(1017)

Amplify by

PCR and

mutagenize

Re-select to

optimize

FINISH:

Optimized enzyme,

therapeutic, or sensor

Figure 5. Teaching an ancient dog new tricks by harnessingnature’s techniques: In vitro evolution of RNA. Starting from alibrary of many diverse molecules, the fittest ones for a certainfunction are selected, amplified, mutagenised, and re-selected untilthe winners fulfil a given task to perfection.

Biologist (2002) 49 (5) 203

be cleverer than that, and only a limited number of enzy-matic activities have been identified to date (Wilson &Szostak, 1999). Again, the predominant classes are thosethat synthesise, chemically modify, or cut nucleic acids,important contributions for supporting the RNA Worldhypothesis. In addition, classes of ribozymes with unre-lated enzymatic activities have been selected. Oneimportant demonstration has been the discovery of apeptidyl transferase enzyme (catalysing the reaction inFigure 1D). This is particularly interesting because thenew enzyme does not necessarily mimic the ribosome’sactive site. The ability to catalyse other reactions, e.g.,alkylations, isomerisations, Diels-Alder cycloadditions,and metal transfers, has also been found (for some exam-ples see Figure 1). This diversity of reaction typesfurther encourages belief in a period of early molecularevolution on Earth where nucleic acids catalysed the fullrange of reactions necessary for their own reproductionand metabolism.

Although many of these nucleic acid enzymes are not asefficient as known protein counterparts, it is not clear thatin vitro evolution of the individual enzymes has beenpushed to its fullest efficiency yet, especially consideringpossible inclusion of cofactors. The beauty of this form ofmolecular evolution is that it can be pushed quite ruth-lessly to attain the goals of the experimentalist.Conditions and accessory molecules can be varied whilemutagenising heavily and applying stringent selection forthe desired function. It is a form of evolution where it ispossible to control all the rules (short of changing physicallaws) and focus on a single outcome. The future of nucleicacid catalysts (and aptamers) therefore seems not limitedto enzymes that might once have existed in an RNA World,but can include solutions to problems not encountered innature. Scientists have already started to fathom theiroptions by selecting high-affinity and -specificity aptamers,which can be used as therapeutics to block function of theirtargets in vivo or as diagnostic sensors to detect them invitro. These applications promise a rich future for today’sexplorers of the RNA World.

Further readingAltman S (1990) Nobel lecture. Enzymatic cleavage of RNA by

RNA. Biosci Rep. 10, 317 – 337.Butcher S E (2001) Structure and function of the small ribozymes.

Curr Opin Struct Biol. 11, 315 – 320.Cech T R (1990) Nobel lecture. Self-splicing and enzymatic activ-

ity of an intervening sequence RNA from Tetrahymena. Biosci

Rep, 10, 239 – 261.Cech T R (2000). The ribosome is a ribozyme. Science, 289,

878 – 879.Gesteland R F, Cech T R and Atkins J F E (1999) The RNA World,

Second Edition. Cold Spring Harbor Laboratory Press, NewYork.

Lewin A S and Hauswirth W W (2001) Ribozyme gene therapy:applications for molecular medicine. Trends Mol Med, 7,221 – 228.

Moore P B and Steitz T A (2002) The involvement of RNA in ribo-some function. Nature, 418, 229 – 235.

Nissen P, Hansen J, Ban N, Moore P B and Steitz TA (2000) Thestructural basis of ribosome activity in peptide bond synthesis.Science, 289, 920 – 930.

Wilson D S and Szostak J W (1999) In vitro selection of functionalnucleic acids. Annu Rev Biochem, 68, 611 – 647.

Websitewww.imb-jena.de/RNA.html This ‘RNA World Website’ is maintained at the Institute for MolecularBiotechnology in Jena and has links to numerous databases, webtools, tutorials and scientific meetings revolving around RNA.

After training for his PhD from 1992 to 1995 with the 1967Chemistry Nobel Laureate Manfred Eigen at the Max-Planck-Institute for Biophysical Chemistry (Göttingen, Germany) andas a postdoctoral fellow from 1995 to 1999 with John M Burkeat the University of Vermont (Burlington, VT, USA), Nils GWalter is now Assistant Professor of Chemistry at theUniversity of Michigan (Ann Arbor, MI, USA). His researchinterests are in the area of structure and dynamics of smallcatalytic RNA, using biophysical tools such as fluorescencespectroscopy. David Engelke is a Professor of BiologicalChemistry at the University of Michigan and an AssociateEditor of the journal of the international RNA Society, RNA.His laboratory investigates the biosynthesis and functions ofsmall RNAs in eukaryotic nuclei.Nils G WalterRm 4821, 930 North UniversityThe University of MichiganAnn Arbor, MI 48109-1055, USAnwalter@umich.eduDavid R Engelke3200 MSRB III1150 W. Medical Center DriveAnn Arbor, MI 48109-0606, USAengelke@umich.edu

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