Zurich Open Repository andArchiveUniversity of ZurichMain LibraryStrickhofstrasse 39CH-8057 Zurichwww.zora.uzh.ch

Year: 2005

Structure Determination of Catalytic RNAs and Investigations of TheirMetal Ion-Binding Properties

Erat, M C ; Sigel, Roland K O

Abstract: Naturally occurring RNA molecules exhibit many unexpected and fascinating properties inliving cells like protein synthesis and transport, regulation of metabolic functions, and catalytic cleavagereactions. To understand this functional diversity, a detailed knowledge of RNA structure and metalion-binding properties is crucial. In our research group, we address these problems by combining variousbiochemical, analytical and spectroscopic techniques. A large part of our work is devoted to the structuredetermination of catalytic RNA molecules, i.e. ribozymes, by NMR. Based on the three-dimensionalstructure, further experiments are carried out to understand in detail the effects of different metal ionson the local and global structure, as well as catalysis itself.

DOI: https://doi.org/10.2533/000942905777675534

Posted at the Zurich Open Repository and Archive, University of ZurichZORA URL: https://doi.org/10.5167/uzh-79691Journal ArticlePublished Version

Originally published at:Erat, M C; Sigel, Roland K O (2005). Structure Determination of Catalytic RNAs and Investigations ofTheir Metal Ion-Binding Properties. CHIMIA International Journal for Chemistry, 59(11):817-821.DOI: https://doi.org/10.2533/000942905777675534

MODIFIEDOLIGONUCLEOTIDES 817CHIMIA2005,59,No.11

Chimia 59 (2005) 817–821 © Schweizerische Chemische Gesellschaft

ISSN 0009–4293

Structure Determination of Catalytic RNAs and Investigations of Their Metal Ion-Binding Properties

MichèleC.EratandRolandK.O.Sigel*

Abstract: NaturallyoccurringRNAmoleculesexhibitmanyunexpectedandfascinatingpropertiesin livingcellssuchasproteinsynthesisandtransport,regulationofmetabolicfunctions,andcatalyticcleavagereactions.Toun-derstandthisfunctionaldiversity,adetailedknowledgeofRNAstructureandmetalion-bindingpropertiesiscrucial.Inourresearchgroup,weaddresstheseproblemsbycombiningvariousbiochemical,analyticalandspectrosco-pictechniques.AlargepartofourworkisdevotedtothestructuredeterminationofcatalyticRNAmolecules,i.e.ribozymes,byNMR.Basedonthethree-dimensionalstructure,furtherexperimentsarecarriedouttounderstandindetailtheeffectsofdifferentmetalionsonthelocalandglobalstructure,aswellascatalysisitself.

Keywords:Affinityconstants·Metalions·NMR·Ribozymes·RNAstructure

removal of non-coding intron sequences,theregulationofmetabolic functions,andeven the regulationof thecellcycle itself[1].SomeoftheseRNAsarenotonly‘pas-sively’ exhibiting their function by ‘just’bindingtootherbiomolecules,butperformalsocatalyticreactions.Theseso-calledri-bozymeswerediscoverednearly25yearsago [2][3]. Nowadays eleven differentclasses of naturally occurring ribozymesareknown, twoof themhavingbeendis-coveredonlylastyear[4][5].

Ourresearchconcentratesontwoclass-esofthenaturallyoccurringRNAs:(i)ribo-switchesand(ii)groupIIintronribozymes.Theemphasisofthisshortreviewisonourstructural and analytical NMR studies ongroup II intron ribozymes;our riboswitchworkisonlybrieflyindicatedinthefollow-ingSection.

2.Riboswitches

Riboswitches are highly conservedRNA sequences occurring for example inthe5ʹ-UTR (seeAbbreviations)of certainmRNAs.TheybindsmallmetaboliteslikeFMN, SAM, TPP, guanine, adenine, andcoenzymeB12withahighdegreeofaffinityandspecificity[6][7].Uponbindingofthecorresponding metabolite, the riboswitchsequencepresumablyundergoesstructuralchanges[8]andinsomecasesevencleav-age of the RNA strand occurs [4]. Bothcleavage and structural changes inhibitbindingofthe5ʹ-UTRtotheribosome,and

thusprohibittranslationtothecorrespond-ingprotein.Astheprotein(s)expressedbyamRNAisalwaysinvolvedineitherbio-synthesisortransportofthemetabolitethatbinds to the riboswitch, this new class ofRNAmoleculesconstitutesanelegantwayoffeedbackcontrolwithinlivingcells.

Inourgroup,weworkwiththeso-calledB12riboswitch.Thisriboswitchconsistsofanabout200ntlongandhighlyconservedsequencelocatedinthe5ʹ-UTRofmRNAsassociatedwiththemetabolismandtrans-port of coenzyme B12 and vitamin B12,respectively[9].Ourinvestigationsencom-pass structural studies as well as bindingstudiesofcoenzymeB12anditsderivativestothisriboswitchsequenceinthepresenceofdifferentmetalions.

3.GroupIIIntronRibozymes

The main efforts of our research arepresently devoted to group II intron ribo-zymes.Theselargemolecularmachinesareself-splicingintrons,i.e.capableofcuttingthemselvesoutof theprecursorRNAandinasecondstepjoiningtheadjacentexonstoyieldthefinalmRNA–allwithout theaidofproteins (Fig.1). Inaddition, theseamazing molecules can insert themselvesagainintoRNAandevenDNA.Theoccur-rence,reactionmechanisms,andthediffer-entaspectsofmetalionbindingtogroupIIintronshavelatelybeenreviewed[10][11],thusonlyafewimportantpointsaresum-marizedbelow:

*Correspondence:Prof.Dr.R.K.O.SigelUniversityofZürichInstituteofInorganicChemistryWinterthurerstrasse190CH-8057ZürichTel.:+41446354652Fax:+41446356802E-Mail:roland.sigel@aci.unizh.chhttp://www.aci.unizh.ch/rna/

1.Introduction

Theresearch inour laboratoryfocusesontheinteractionofmetalionswithRNA,i.e.whichrolesdifferentMn+haveonfolding,structure, andcatalyticpropertiesof ribo-nucleicacids.LikeDNA,RNAisapoly-anionunderphysiologicalconditions,witheverybridgingphosphategroupaddingonenegativechargetotheoverallcharge.Itfol-lowsthatnucleicacidscannotoccurwith-outmetalionsbeingpresent.RNAfulfillsmanyfunctionsinalllivingorganismsandnew roles of these fascinating moleculesarediscoveredeveryyear.ThebestknownRNAsarecertainlymRNAandtRNA,butalsotheribosomeitselfismainlycomposedofRNA.

Other functions carried out by RNAsincludeprotein transport, splicing, i.e. the

MODIFIEDOLIGONUCLEOTIDES 818CHIMIA2005,59,No.11

3.1.GeneralRemarksonGroupIIIntronRibozymes

GroupIIintronsaredefined(i)bytheirsecondary structure consisting of six do-mains radiatingout froma centralwheel,(ii)byasetofhighlyconservednucleotidesand(iii)bytertiarycontactsspreadthrough-out thewhole intron (Fig.2).Thesecata-lyticRNAsarefoundinorganellargenesofplants,fungiandlowereukaryotesaswellas inbacteria [10].SomegroupII intronscontain an ORF encoding for a maturaseprotein with a reverse transcriptase and anucleotideexcisiondomain[10].Togetherwiththematurase,theseintronsactasmo-bilegeneticelementsthatcaninsertthem-selves intoDNAathomologousandevenheterologoussites.

Basedonphylogeny,groupIIintronsarethoughttobeveryoldandtohaveplayedanimportantroleineukaryoticevolution[10]:Includingnuclearintrons,thetransposableLINEelementsandspliceosomalRNA,asmuchas30%ofthehumangenomemightstemfromancestralgroupII introns [12].Group II introns still show a rather closesequence relationship to the spliceosome,

theeukaryoticsplicingmachinerythatcon-sistsoffiveRNAsand>100proteinsandisresponsiblefortheaccurateremovalofallintrons. It is surprising to see thatdespitetheir evident importance in evolution andtheirdirectroleinthecorrectexpressionofproteinsinmanyorganisms,relativelylittleisknownabout thestructureandcatalyticmechanismsofgroupIIintronribozymes.

To perform the self-splicing reaction,groupIIintronsfoldintoacompactthree-dimensional structure. Out of the six do-mains,onlydomains1,5,and6(D1,D5,D6)togetherwiththelinkerregionsarees-sential for the complete splicing reaction:D1bindstheexonsequencesandprovidesthescaffoldforthethree-dimensionalstruc-ture,D5 isviewedas thecatalyticcenter,andD6harbors thebranchadenosine, the2ʹ-OHofwhich servesas thenucleophileinthefirststepofsplicing(Fig.1).Toper-formcatalysis,groupIIintronshaveaveryspecific need for metal ions as cofactors.Monovalent (usually Na+ or K+) as wellas divalent metal ions (usually Mg2+) areintrinsically tied to all RNAs for chargecompensation.Withoutmetalions,thehigh

negative charge of the sugar-phosphatebackbone would prevent any higher-orderfoldingofRNAmolecules.Inaddition,atleastforthelargerribozymeslikethegroupII introns, more and more experimentalevidencesuggestsadirect involvementofdivalent metal ions in catalysis [11]. ThelittlethatisknownaboutmetalionbindingingroupIIintronshasbeenreviewed[11]and is therefore only briefly described inthenextSection.

3.2.GeneralAspectsofMetalIonBindingtoRNA

Metal ion binding to RNA moleculesdiffersconsiderablyfrombindingofmetalionsinmetalloproteins[13]:(i) Generally, proteins do not carry a

large charge. Some domains may becharged, but folding to the correctthree-dimensional structure is pre-dominantly determined by a hydro-phobic collapse. In contrast, RNAsare highly negatively charged, asthey contain one phosphodiester pernucleotide.Itfollowsthatfoldingcanonlyoccurinthepresenceofsuitablecounterions,whicharepredominantlyalkalineionsaswellasMg2+asthesearemostabundantinlivingcells.

(ii) Proteins consist of 20 amino acids.Among them, only four (Cys, Asp,Glu,His)comprisethemajorityofallmetalionbindingsites.Incontrast,ne-glectingallpost-transcriptionalmodi-fications, RNA consists of only fournucleotides, each of which containsseveral possible metal ion-coordinat-ing sites. RNA-metal ion binding isusually weak, and as a consequenceoften poorly defined. Nevertheless,RNAcanachieveasurprisinglyhighspecificitywithrespecttothesitesofmetalcoordinationaswellasthekindof metal ion involved: For example,small amounts of Ca2+ efficientlycompete with Mg2+ ions and inhibitthesplicingreactionofthegroupIIin-tronai5γininvitrosplicing[14][15].

(iii)Depending on their metabolic func-tion,proteinsbindandusealargeva-riety of different metal ions, rangingfromalkalineandalkalineearthmetalions to numerous d10 and transitionmetal ions [16]. In contrast, in natu-rally occurring RNAs, only Na+, K+

andMg2+havesofarbeenidentifiedasnaturalcofactors.

(iv)Mg2+isakineticallylabilemetalion.Thus,ifanexcessofpossiblecoordi-natingatomsispresent,Mg2+maynotbefixedtothesameatomsatalltimes.In addition, inner-sphere and outer-sphere,i.e.directandwater-mediatedbindingispossible.

Taken together, the four points men-tionedaboveleadtothefollowingtwoma-

Fig.1.Splicingofgroup II intron ribozymes.The intron isdepictedasablack line incorporating a highly conserved adenosine (A), the 2ʹ-OHofwhichactsas thenucleophile in the firststepofsplicing.Thetwoexonpiecesflankingtheintronareshownasgreyboxesthatarejoinedtogetherduringthesplicingprocess.Bothstepsarereversible.

Fig.2.SecondarystructureofgroupII introns.TheconservedsecondarystructureofthegroupIIintronsai5γ found in the cox1 gene of S. cerevisiae (A) and Pl.lsu/2 located in the large ribosomal subunit of theAtlanticbrownalgaeP.littoralis(B)areshown.Sixdomains(D1–6)radiateoutfromacentralwheel.Uponfoldingtotheactivestructuremanytertiarycontactsareformed,whichareindicatedbyGreekletters.The5ʹ-splicesite ismarkedwithanasteriskandisdefineduponbasepairingoftwointronicsequencesEBS1andEBS2(exonbindingsite)withthecomplementaryexonicIBS1andIBS2(intronbindingsite)sequences.

MODIFIEDOLIGONUCLEOTIDES 819CHIMIA2005,59,No.11

jorissuesforthedetectionofM2+bindingsitesinRNA:(a)Howtodetectthefewspe-cificbindingsiteswithinanoceanofunspe-cificallyboundionsofthesamekind?(b)HowtodetectK+orMg2+,bothofwhicharespectroscopicallysilent?

Weapplyacombinedapproachofmeth-odsfromdifferentresearchfieldstoaddressand answer these questions regarding theroleofmetalionsinstructureandfoldingofRNAs[15][17–19].

4.NMRStructureDetermination

Inorder to investigatemetal ionbind-ing to RNAs, structures at atomic resolu-tionareneeded.Wefollowadualapproachtoachievethischallenginggoal,onebeingX-ray crystallography (in collaborationwith Dr. Eva Freisinger in our Institute)and the other NMR spectroscopy. NMRspectroscopy is a powerful tool to studythestructure, tertiaryinteractions,dynam-ics and requirements for metal ions ofRNAmolecules.Usually,RNAsof a sizebetween20and40nucleotidesarestudiedbyNMR.Nevertheless,recentlya30kDadimeric RNA structure (86 nt) has beensolvedathighresolutionsettingthefuturedirectionofNMRstructuredeterminationsofnucleicacids[20].Thefactthatthissizecorrespondstoonlyaboutonethirdofwhatisachievedwithproteinsnowadayscanbeattributed to the small number of protonspresent,tothelackindiversityofresiduesand consequently to the heavy overlap ofresonances(Fig.3A).

AsanentiregroupIIintronistoolargetobestructurallyexaminedbyNMR,wein-vestigateindividuallythedifferentdomainsinvolvedincatalysis[17][19].First,thede-siredRNAissynthesizedinlargeamountsbyinvitrotranscription,asdescribedinourother contribution in this CHIMIA issue[21]. For a complete structure determina-tion by NMR, several samples of 0.5–1mM RNA in 220 μl 100% D2O or 90%H2O/10% D2O are needed (both naturalabundanceaswellas isotopically13C,15NenrichedRNA).

First, the best conditions for obtain-ing high-quality NMR spectra need to bedetermined by optimizing factors such astheRNAsequence,temperatureandpHaswell as concentrationsofmonovalent andpossiblydivalentmetalions.AlsotheRNAconcentration can be crucial, because ontheonehand,higherconcentrationsleadtoshortermeasurementtimes,butontheotherhandtheycanalsoleadtodimerizationofahairpinsequence,givingadouble-strandedstructure.Dimerscanoftenberemovedbygelfiltration[21]orcircumventedbydilu-tionof theRNAintoa largevolume(e.g.100ml)ofwater,followedbyheatingto95°Cforuptoonehourandsubsequentslow

cooling to room temperature and concen-trationoftheRNAbyultrafiltration.Inthisinitial phase of a structure determinationmainly 1D spectra in D2O and 90% H2Oarerecordedtoobtainafirstindicationonthesamplequality.

Once optimized conditions are found,2D1H,1H-NOESYspectrainD2O(Fig.3)and90%H2Oarerecorded.InthesespectraNuclearOverhausercrosspeaks(NOE)are

observedbetweenprotonsthatarecloserinspace than 5–6 Å, yielding distance con-straints that are subsequently needed forstructurecalculation.Akeystepinassign-ingallresonancesistheso-called‘sequen-tialwalk’(Fig.3B):Thearomaticnucleo-base-protonsarecloseinspacenotonlytotheir intra-residue ribose H1ʹ, but also totheH1ʹoftheupstream5ʹ-nucleotide(Fig.3C).Thus, theconnectivityofnucleotidesalong a RNA strand can be established.Several recent reviews give a good over-view on the exact methodology of reso-nanceassignmentsforRNAs[22–26].Forfullassignmentofallpeaksandverificationthereof,aseriesof2D1H,1H-NOESYspec-trainD2OaswellasinH2Oarerecordedatdifferenttemperaturesandmixingtimes.Other special multidimensional NMR ex-periments give further information: JHNN-COSYexperimentsrevealtheexistenceofhydrogenbonds,residualdipolarcouplings(RDCs)yieldorientational restraints [27],and HCCH-COSY/TOCSY experimentsareneededtoidentifythespinsystemwith-inthesugarresidues.Forthelatterexperi-ments,13C,15N-enrichedRNAisrequired.AttheUniversityofZürich,weareintheluckyposition tohaveaccess toanexcel-lent NMR facility with high-field NMRmachinesrangingfrom500–700MHz,in-cludingcryoprobes toenhancesensitivity,enabling us to take advantage of the fulldiversityofpossibleexperiments.

Once a set of distance and conforma-tional restraints iscollected,structurecal-culationisstartedfromaninitialstructurebyafullycomputationalsimulatedanneal-ingprotocol(X-PLORNIHpackage[28]),yieldingafirstsetofstructures.Theseout-putstructuresareanalyzedfordistanceandconformational violations, the input filescorrected and supplemented with furtherrestraints,beforeanextroundofsimulatedannealingisperformed(Scheme).Thiscy-cleisrepeateduntilatleast10%oftheout-putstructuresconverge(e.g.20outof200calculated structures) [17][19]. Usually, ar.m.s.d.of1–3Åforallheavyatomsofsuchan ensemble of 20 structures is obtained.Thisr.m.s.d.islargerthanforcomparableprotein structures,butone shouldkeep inmindthatRNAisnotascompactasapro-teinandthusmuchmoreflexible.

Afterthedeterminationofthethree-di-mensionalstructureofaRNAmolecule,wefocusonitsbindingpropertiestometalionsandtheireffectonlocalstructures.

5.InvestigationofMn+BindingtoRNA

Asmentionedabove,themetalionsusu-allyassociatedwithnucleicacids(Na+,K+,andMg2+)arespectroscopicallysilentandthusdifficulttodetect.Nevertheless,there

Fig. 3. Structure determination of RNAs byNMR. A) Typical 1H,1H-NOESY spectrum ofaRNAwith an evident small dispersion of theresonances clustering in specific regions. B)SectionofthespectrumframedinA)withtheso-called‘sequentialwalk’indicatedasablackline,connecting all nucleotides sequentially along ahelical RNA. C) Through-space connectivitiesof nucleotide protons along a RNA sequenceleadingtothe‘sequentialwalk’.

MODIFIEDOLIGONUCLEOTIDES 820CHIMIA2005,59,No.11

areseveralwaystoexaminemetalionbind-ingtoRNA.RecentlywehaveshownthatgroupIIintronspossessnumerousspecificMn+bindingsiteswithinthecatalyticcoreofthefoldedRNAmoleculebypartiallyre-placingMg2+withTb(III)[18][29].Tb(III)replacesMg2+atitsbindingsitesandleadsto a hydrolytic cleavage of the phosphatesugarbackbone,whenboundintheminorgroove. If Mg2+ is replaced with Fe2+/3+,cleavage occurs through a radical mecha-nism(‘Fentoncleavage’)andthusirrespec-tiveofthebindingsite[30].

Afurtherwaytodetectmetalionbind-ing are so-called thio-rescue experiments.Here, one of the non-bridging phosphateoxygens is replaced by a sulfur. If Mg2+

bindingtothissiteisessentialforcatalysis(orfolding),theactivityoftheribozymeisreduceddue to the loweraffinityofMg2+towardsthesoftersulfurligand[31].UponadditionofmorethiophilicmetalionslikeCd2+orZn2+,whichcoordinatereadilytosulfur, ribozyme activity may be restored[32]. Indeed, such experiments are notunequivocal and often rescue is observedalsowithmetal ions that arehardlymorethiophilicthanMg2+,likeMn2+,butwhichhaveahigheroverallaffinity[31].TogainabetterunderstandingontheeffectofsulfurwithinRNAoligonucleotidesonstructure,hydrogenbonding,andmetalionbinding,weperformNMRstudiesonsuchmodifiedRNAs[33].

NMRspectroscopyenablesustodirect-lymonitorMg2+bindingtoRNA[17][19].Additionof increasingamountsofMgCl2to a RNA solution leads to changes inchemical shifts of specific protons (Fig.4B).ThesechangescaneitherbecausedbydirectMg2+bindingataneighboringatom,orbyaslightstructuralchangeinlocalge-ometryuponMg2+bindingnearby.Byfit-tingtheexperimentaldatatoa1:1bindingmodel,affinityconstantsforMg2+bindingcanbecalculated[17][19].WecouldshowthatRNAhairpinslikeD5[29]orD6[17]bind3–5Mg2+ionsatspecificsiteswitharatherhighaffinity. Interestingly, someofthesesitesareveryclosetothecatalyticallyimportantnucleotides implyingapossiblecatalyticroleoftheseions.Consideringthechargerepulsionbetweenmetalionscloseinspace,itisremarkablethatnucleicacidshavetheabilitytobindmetalionstoseveralsitesincloseproximity,aswecouldshowbypotentiometricpHtitrations[34].

Metal ions not only bind passively toRNAbutcanleadtoasubstantialdecreasein pKa values of nucleotide functionalgroups [35], a change in hydrogen bond-ingproperties[35],orperhapsevenastruc-turalchange.We investigate thesevariouseffectsbyfluorescencespectroscopy[36],Scheme.

Fig.4.Solutionstructuresandmetal ionbindingpropertiesofcatalyticdomainsofgroupII intronribozymes.A)SchematicsecondarystructureofagroupIIintrontogetherwiththesolutionstructuresofD5[19]andpartofD6[17],assolvedinourlaboratory(PDBcodes1R2Pand2AHT).B)ChangeofthechemicalshiftofaH2protonwithinD6upontitrationwithMgCl2.Theexperimentaldataisfittoa1:1bindingmodeltogivetheaffinityconstantKA=112±26M

–1(logKA=2.05±0.10).

MODIFIEDOLIGONUCLEOTIDES 821CHIMIA2005,59,No.11

biochemical activity assays [14][15], po-tentiometric pH titrations [37], as well asbyNMR[17][19].Byapplying thiscom-bined approach we make use of the indi-vidualadvantages thateachof these tech-niquesoffers.Ideally,wetherebyachieveafullcharacterizationoftheroleofspecificmetalionsinthestructureandfunctionofourRNA.

6.ConcludingRemarks

In contrast to the traditional view ofribonucleicacidsasmereworkingcopiesofthegenome,rapidlyincreasingexperi-mentalevidenceattributesa largevarietyofspecificcellularfunctionstoRNA,in-cluding protein transport and synthesis,metabolic regulation, and even catalyticreactions [1]. The polyanionic characterof RNA makes charge compensation in-dispensable.Thisisusuallyaccomplishedbyweakandunspecificbindingofmono-anddivalentmetalionstothephosphate–sugarbackbone.Ontheotherhand,RNAemploys distinct metal ions to stabilizehigherorderstructuresandtoactdirectlyascofactorsinRNA-catalysis[13].Thesemetalions,usuallyMg2+,bindwithanex-ceptionallyhighdegreeofspecificityandaffinitytotheirassignedsites.

Asindicatedabove,theresearchinourlaboratory focuseson the characterizationofthesemetalionsandtheirrolesinfold-ingandfunctionintwospecificclassesofnaturally occurring RNAs: riboswitchesandgroupIIintronribozymes.Thisreviewsummarizesour recentNMRstudieswiththegroupIIintronribozymeai5γ.Wehavesolved the solution structures of domain5 (D5) [19] comprising the catalytic cen-ter as well as of a shortened construct ofthebranch-pointdomain6(D6)[17].Thepreceding pages provide an overview ofthedifferentstepsthatleadtothestructuredetermination of these two RNA hairpinsbyNMR.

Subsequent to the structure determina-tion,wearestudyingtheeffectofdifferentdivalentmetal ionsonthesetwoimportantdomainsandtheirability tocarryout theirfunctionaswellastoinvolvethemselvesintertiary interactionswithotherpartsof theintron.Tothisend,weapplyawiderangeoftechniques,includingNMR,X-raycrys-tallography,fluorescence,UVandCDspec-troscopy,biochemicalassaysandpotentio-metricpH-titrationstogaininsightintothediverserolesthatmetalionscanplayinRNA

structure and function.With our work, wehopetoshedlightontheroleofmetalionsinRNAandtoemphasizethenecessitytoin-tegratethefieldsofchemistryandstructuralbiologyforgainingabetterunderstandingofthesefascinatingmolecules.

AbbreviationsAsp,asparticacid;Cys,cysteine;EBS,

exon binding site; FMN, flavin mononu-cleotide;Glu,glutamicacid;His,histidine;IBS,intronbindingsite;mRNA,messengerRNA; ORF, open reading frame; r.m.s.d.,root mean square deviation; SAM, S-adenosylmethionine; TPP, thiamine pyro-phosphate; tRNA, transfer RNA; 5ʹ-UTR,5ʹ-untranslatedregion.

AcknowledgementsWe thank Dr. Eva Freisinger for careful

reading, helpful comments and discussionsregarding this manuscript. R.K.O.S. thanks alltheenthusiasticmembersofhisresearchgroup:MichèleErat,MayaFurler,SofiaGallo,Dr.BerndKnobloch, Daniela Kruschel, Miriam Steiner,and Veronica Zelenay for their devoted workand valuable contributions. Financial supportby the Swiss National Science Foundation(SNF-Förderungsprofessur PP-002-68733/1) isgratefullyacknowledged.

Received:August23,2005

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