Comp Structural Bioinfo
Comp Structural Bioinfo ECS 129
Popular in Course
Popular in Engineering Computer Science
This 21 page Class Notes was uploaded by Ashleigh Dare on Tuesday September 8, 2015. The Class Notes belongs to ECS 129 at University of California - Davis taught by Staff in Fall. Since its upload, it has received 50 views. For similar materials see /class/191732/ecs-129-university-of-california-davis in Engineering Computer Science at University of California - Davis.
Reviews for Comp Structural Bioinfo
Report this Material
What is Karma?
Karma is the currency of StudySoup.
Date Created: 09/08/15
Article No jmbi19993312 available online at httpwwwidealibrarycom on Inigl i J Mol Biol 1999 294 829 849 REVIEW ARTICLE Stitching Together RNA Tertiary Architectures Thomas Hermann and Dinshaw J Patel Cellular Biochemistry and Biophysics Program Memorial SloanKettering Cancer Center New York NY 10021 USA The powerful explanatory paradigm of molecular biology requiring form to coevolve with function has again been proven successful when over the recent two decades a wealth of biological functions have been uncov ered for RNA Previously considered as a mere mediator of the genetic code RNA is now acknowledged as a key player in a wide variety of cel lular processes Along with the discovery of novel biological functions of RNA molecules a number of RNA threedimensional structures have been solved which beautifully demonstrate the molecular adaptability which allows RNA to participate as a key player in these functions A distinct repertoire of molecular motifs provides a basis for the assem bly of complex RNA tertiary architectures 1999 Academic Press Keywords metal ions and hydration ribozymes and catalysis RNA Corresponaling authors folding and function RNA motifs X ray NMR and molecular modeling Introduction The finding of numerous key cellular processes associated with RNA molecules asx active players has changed our view on nucleic acids and it is now acknowledged that the diversity of RNA func tionality is on a par with that of proteins reviewed by Gesteland amp Atkins 1993 Gesteland et al 1999 RNA is no longer considered solely as a pas sive transporter of the genetic code but as an extremely versatile class of molecules actively par ticipating in all steps of gene expression RNAs that specifically recognize substrate molecules eg aptamers reviewed by Famulok 1999 Patel 1997 and catalyze chemical reactions eg ribozymes reviewed by Carola amp Eckstein 1999 Lilley 1999 McKay amp Wedekind 1999 have been discovered both in nature and by in vitro selection The specific functions of RNA molecules are modulated by their distinct threedimensional structures While for over two decades regular helices of doublestranded RNA and the transfer RNA tRNA fold had been the only known examples of RNA threedimensional structures reviewed by Saenger 1984 the last years have seen an explosion of novel RNA architectures Technical improvements in RNA s thesis and structure determination methods have led to the solution of many threedimensional structures of RNA molecules and complexes between RNA and both proteins and small molecules Especially Email addresses of the corresponding authors thermannsbnmr1skimskccorg pateldmskccorg 00222836 99 490829 21 3000 0 the crystal structure of the P4P6 domain of self splicing group I introns Cate et al 1996a has revealed a treasure of novel RNA interaction motifs Batey et al 1999 Even before detailed structural information on RNAs other than tRNA was available the domi nance of hierarchical principles in RNA folding Brion amp Westhof 1997 has allowed the construc tion of large RNA structural models Michel amp Westhof 1990 Lehnert et al 1996 which agree extremely well with the available experimental data Golden et al 1998 With a growing number of threedimensional structures of RNA molecules at hand reviewed by Masquida amp Westhof 1999 Nowakowski amp Tinoco 1999 FerreD Amare amp Doudna 1999 we have begun to understand the principles governing the architecture of RNA folds Here we discuss basic motifs of RNA structure involved in stabiliz ing RNA tertiary folds The paradigm of RNA architectures building handles into helices In RNA folds usually more than half of all nucleotides participate in standard WatsonCrick basepairing Consecutive stacking of such canoni cal basepairs gives rise to Aform helices of double stranded RNA Close packing of doublestranded helices is the principle governing the basic architec ture of all higherorder RNA folds which have been structurally elucidated so far Figure 1 Instead of major and minor grooves as in Bform doublestranded DNA Aform helices have charac 1999 Academic Press Stitching Together RNA Tertiary Architectures teristic deep and shallow grooves Since the discri minatory edges of the bases are buried in the deep groove regular Aform helices exert little potential for specific interactions with other domains Weeks amp Crothers 1993 In order to build the intricate threedimensional architectures observed in RNA folds Westhof amp Michel 1998 Ferr D39Amar amp Doudna 1999 specific interaction sites are grafted into helices Such sites comprise structurally con served modules which have been found in the threedimensional architectures of many different RNA molecules Among these molecular modules are 1 variations of the WatsonCrick basepairing scheme ie mismatches 2 triples and quadruples of interacting bases 3 platforms with pairing between consecutive bases within one strand 4 bulgedout residues 5 alternate crossstacking between bases in different strands ie interdigita tionquot and 6 recurring hydrogenbonding pattern between riboses of consecutive nucleotides in two strands ie ribose zipperquot Interactions in the plane basepairing in mismatches triples quadruples and platforms Hydrogenbond interactions between sets of coplanar bases allow for a large number of mis match base combinations beyond the canonical AU GzC WatsonCrick and GU wobble pairs Gautheret amp Gutell 1997 More than 25 years ago the threedimensional structure of tRNA the first and most concisely analyzed RNA fold revealed the geometry of a few noncanonical base interactions Robertus et nl 1974 Kim et 51 1974 Figure 1 The arrangement of doublestranded helices governs the overall architecture of RNA folds The threedimensional struc tures of a number of complex RNA molecules are shown here in order to illustrate the packing of helices along with the intricate folding observed in RNA architectures a The P4P6 domain of the self splicing group I intron from Tetra hymenn thermophiln Cate et ML 1996a b The hammerhead ribozyme Pley et ML 1994a Scott et ML 1995 c The genomic ribo zyme from hepatitis delta virus HDV Ferr D Amar et ML 1998 d PhenylalaninetRNA from yeast Robertus et ML 1974 Kim et ML 1974 e The L11binding domain of 23 S rRNA from Escherichia coli bound to the L11 protein protein not shown Conn et ML 1999 Wimberly et ML 1999 Since then mismatch pairs have been found widely in RNA architectures Figure 2 Inserted into RNA helices mismatch pairs par ticipate in stacking interactions Holbrook et ML 1991 and provide recognition sites both by pre senting functional groups at the base edges and through distortion of the regular helical backbone In the loop E of 5 S ribosomal RNA rRNA four purinepurine basepairs interspersed in a regular Aform helix give rise to a widened deep groove and a unique hydrogenbonding surface in the shallow groove permitting recognition by riboso mal proteins Figure 2b Szewczak et 51L 1993 Szewczak amp Moore 1995 Correll et 51L 1997 Dallas amp Moore 1997 The extensive mismatch pairing in loop E is stabilized by metal ions and organized water molecules Correll et nl 1997 indicative of cations and solvent involvement in the maintenance of noncanonical basepairs see below Leontis amp Westhof 1998a The loop E module itself has been found as a conserved motif in ribosomal RNAs RNase P the hairpin ribozyme and several selfsplicing introns Leontis amp Westhof 1998b Among homopurine basepairs G A mismatches stand out as they are the most common noncano nical structural motifs in RNA molecules Gautheret et 51L 1994 The sheared configuration of GA pairs has been found as a conserved build ing block in the threedimensional structures of many RNAs Heus et 51L 1997 among them the GNRA loops Heus amp Pardi 1991 N is any nucleotide R is a purine see below the GNRA Stitching Together RNA Tertiary Architectures 831 2 b substrates as in the theophylline aptarner RNA Zirnrner magenta U green c are marked by color coding as follows A orange lt3 i is in red like pentaloop of boxB RNA in the complexes with peptides Cai at LIL 1998 Legault at LIL 1998b and the architectures of several rib z mes Cate at LI 1996a Pley at LIL 1994a Scott at LIL 1995 Butcher at LIL 1999 and ribosomal RNA fragments Szewczak at LIL 1993 Szewczak amp Moore 1995 Correll at LIL 1998 Conn at LIL 1999 Wimberly at LIL 1999 oubles anded motifs other than Aform helices are created by consecutive stacking of mis match pairs providing open structures as docking surfaces in RNA terti interactions which is illus trated by the loop B of the hairpin ribozyme Figure 2c Butcher at LIL 1999 A peculiar case of consecutively sta d mismatches occurs in the crystal packing of the leaddependent ribozyme Wedekind amp McKay 1999 in which homo purine airs are formed between bases within two paral leloriented strands making up a short parallel helix The variety of partners for mismatch basepair ing is extended by the potential of A and C nucleo bases for pHdependent protonation providing additional hydrogen donor sites as have b Figure 2 a Hydrogensbondjng ractjons between co 1anar bases yield WatsorbCiick pairs the bui e b r t In this and all following Figures bases blue The theophyllirte substrate in 1997 marm 2t LIZ observed for ex 1996 Hoogstraten at at LIL 1998 and G ample in CAquot Cai amp Tinoco LIL 1998 Iang at LIL 1998 Pan A mismatches Fan et LIL WatsonCrick pairs and mismatches can partici pate in triple and quadruple interactions in sets of coplanar bases Base triples have already been foun e ear 1 structures of tRNAs Robertus at LIL 1974 Kim et LIL 1974 Depending topological connection of the strand which provides the third base docking to the pair triples can lead to widened grooves within double stranded architectures or they mediate the tertiary interaction of a third strand An RNA deep groove opened a U U triple is the common theme of the binding sites for arginineamide in the human immunodeficiency virus type 2 HIV2 trans activating region TAR RNA Brodsky amp 39 39 the ovine 39 39 39 Ye at LIL 1995 Puglisi at LIL 1995 and for the HIV 1 Revpeptide in an RNA a tamer Ye at LIL 1996 Docking of adenosine to the shallowgroove edge of a canonical GC pair results in an ACzG triple Stitching Together RNA Tertiary Architectures which has been recurrently observed in strand junctions of RNA folds such as the P4P6 domain of group I ribozymes Cate et al 1996a hepatitis delta virus HDV ribozyme Ferr D Amare et ul 1998 a turnip yellow mosaic virus pseudoknot Kolk et ul 1998 the L11proteinbinding domain of 23 S rRNA Conn et ul 1999 WiInberly et ul 1999 and a ribosomal frameshifting pseudoknot Su et ul 1999 A particular strand junction mediated by triples is the tertiary interaction between a tetraloop and a tetraloop receptor seen in the crystal structures of hammerhead Pley et al 1994b and group I Cate et al 1996a ribozymes Figure 2e The tetraloopreceptor motif first proposed from sequence comparisons and model ing Michel 8 Westhof 1990 is probably the most fundamental building block mediating RNA ter tiary interactions Abramovitz 8 Pyle 1997 see below Stacks of consecutive base triples give rise to tri ple helices Depending on the direction of approach of the third base to the WatsonCrick pair a distinction can be made between major and minor groove respectively deep and shallow groove triplexes A shallow groove RNA triplex comprising six layers of triples has been discovered in the crystal structure of a ribosomal frameshifting pseudoknot Su et ul 1999 providing the first example of an extended triple interaction in the RNA shallow groove Shorter shallow groove tri plexes mediate the tertiary contacts of GNRA tetra loops with receptor duplexes see below Several RNA structures with the third strand in the deep groove are known Klinck et ul 1995 Holland 8 Hoffman 1996 a feature also common to DNA triplexes De los Santos et ul 1989 Rajagopal 8 Feigon 1989 The GAAA tetraloopreceptor motif of the P4P6 domain of group I ribozymes also provides an example of a G A CzG quadruple in which four bases in a plane associate by hydrogen bonding Cate et al 1996a An A A C A quadruple has been proposed to bridge the P4P6 and P3P9 domains of group I ribozymes Golden et ul 1998 Due to the low resolution of the crystal structure of the intact group I ribozyme however a conclusive proof of this quadruple is still lacking The only other quadruple in RNA proven so far is an A C VGzC interaction in the loop of pseudoknots in an HDV ribozyme Ferr D Amar et ul 1998 Hilbers et ul 1998 and a ribosomal frameshifting signal Figure 2f Su et ul 1999 This quadruple is organized by a protonated C which forms hydrogen bonds with each of the three other bases In RNAs the A C VGzC quadruple in the pseudo knots along with C VGzC triples in a mutant TAR RNA Brodsky et ul 1998 and in a tentative RNA triple helix Klinck et ul 1995 are the only examples for mismatches with protonated cytosine bases yet Alignments involving protonated cyto sine bases have been seen before in multistranded DNA structures see for example De los Santos et ul 1989 Rajagopal 8 Feigon 1989 Gehring et ul 1993 Sidebyside basepairing between consecutive nucleotides within one strand of RNA gives rise to platform motifs first observed for vicinal adeno sine bases in the P4P6 domain of group I ribo z es Figure 2g Cate et al 1996b In adenosine platforms the rise of the RNA backbone is reduced thus creating a notch which presents the paired adenosine bases as a stacking surface for other bases Stabilization of adenosine plat forms requires the presence of monovalent metal ions preferably potassium which bind to a specific pocket immediately below the two adenosine bases see below Basu et ul 1998 where in all adeno sine platforms a noncanonical basepair is located One of the three adenosine platforms in the P4 P6 domain participates in the formation of a tetraloop receptor motif see below An adenosine platform similar to the one in the P4P6 domain has been found in the L11binding domain of 23 S rRNA Figure 2h Conn et ul 1999 WiInberly et ul 1999 In a theophylljne aptamer RNA the back bone of consecutive A and C residues adopts the conformation of the adenosine platform giving rise to an AC platform which provides a stacking sur face for the theophylline substrate Figure 2i Zimmermann et ul 1997 This function of the AC latform resembles the role of a G UzA triple serving as the substrate binding surface in an aptamer RNA for avin mononucleotide FMN Fan et ul 1996 Out of the plane crossstrand stacking of bases The stacking interactions between the bases mediated by their nelectron system can be con sidered as the nucleic acids analogue of the hydro phobic core in proteins Stacking of bases contributing significantly to the stability of RNA architectures occurs predominantly between con secutive residues within one strand reviewed by Saenger 1984 In a number of RNA threedimen sional structures crossstrand stacking of bases belonging to different strands is also observed Figure 3ad Again the tRNA fold has provided the first example of crossstrand stacking In the corner of the Lshaped tRNA four interleaved purines participate in the junction of the T and D loops Figure 3c Robertus et ul 1974 Kiln et ul 1974 Recently when other examples of such stacks of interdigitated bases have been discovered in RNA architectures the term base zipper was coined yielding to the apparent need for a taxonomy of RNA structural motifs Base zippers have been found in the structures of a theophylline aptamer RNA Figure 3d Zimmermann et ul 1997 a tet raloop receptor RNA Butcher et ul 1997 and the loop B of the hairpin ribozyme Butcher et ul 1999 Interdigitation contributes significantly to the stability and compactness of the threediluen sional structures of these RNAs with the exception Stitching Together RNA Tertiary Architectures 833 of the tetraloop receptor in which the base zipper forms an open and exposed stretch This howev lish ternary nta rms hydrogen bonds With residues in t e base zipper see below Upon docking of th loop a local conformational rearrang take place in order to convert the base zipper as in the free receptor into an adenosine platform observed in the docked state see below The cross strand staclcin of purines in the base zipper of the ha39 in ribozyme B loop resembles the interactions observed in other RNA archite tures Crossstrand stacked purines occur in the Figure 3 Tight interlocking ot RNA strands is provided by ard crossstrand stacking ot bases and e t ribose zippers a Cross strand stacking o t s ands is o ditterent o ved sometimes eve tor Watsoannck pairs as in tn Col RNA 1oop imer where e guanosine bases o two consecutive canonical cc p g each otner e or Extensive crossstran stacking occurs between two uano bases one ot wnicn participates in sneared GA air in tire sarcin ns rv simi1ar overall structure ot tne two ands which are stitched together sarcinricin loop of eucaryal 28 S rRNA Figure 3b Szewczak at LIL 1993 Szewczak t3 oore 1995 Correll at LIL 1998 the loop E of 5 S rRNA Wimberly at LIL 1993 Correll at LIL 1997 Dallas t3 Moore 1997 the aptamer RNA for FlHI Fan et LIL 1996 the bulgehelixbulge splice site 39ener t3 Moore 1998 and an RNA contain ing the binding site of signal recognition particle Schmitz at LIL 1999 In both ribosomal RNA frag ments one o e guanosine bases involved in th crossstrand stacking participates also in a sheared GA air Even for canonical GC base pairs cross strand stacking between purines is possible exceeding the Stitching Together RNA Tertiary Architectures small purine overlap seen in regular YpR steps where Y is a pyrimidine and R is a purine as has inverted loop RNA dimer Lee 8 Crothers 1998 At the junction of an Aform helix and a loop involved in basepairing with a second RNA monomer the closin GC air of the helix is retracted towards the shallow groove so as to allow crossstrand stacking with an adjacent CG pair in the helix Figure 3a Backbone interactions ribose zippers Regular hydrogenbonding patterns occur between the bases and in some cases also between the backbone riboses in RNA architectures In the densely packed core regions of RNA folds where the backbones of two strands come close ribose zipper structures have been discovered which are held together by hydrogen bonds involving the 2 OH groups of the sugar moieties Figure 3e and f This type of interaction is unique to RNA since DNA lacks 2 OH groups Hydrogen bonding between 2 OH groups of consecutive riboses was first noted in intermo lecular crystal packing contacts of RNA molecules in a hammerhead ribozyme Pley et al 1994b and a doublestranded RNA dodecamer Schindelin et al 1995 Later intramolecular ribose zipper motifs have been found in the threedimensional folds of the P4P6 domain of group I ribozymes Figure 3e Cate et al 1996a the HDV ribozyme Figure 3f Ferr D Amar et al 1998 and the L11binding domain of 23 S rRNA Conn et al 1999 Wimberly et al 1999 A ribose zipper has been suggested connecting the two helical domains of the hairpin ribozyme Earnshaw et nl 1997 In all cases the local geometry of the hydrogen bonding network is conserved despite the dissiini larity of the conformations of the RNA strands which are held together The 2 OH group of the ribose in one strand forms two hydrogen bonds to both the 2 OH and either a pyrimidine OZ atom or a purine N3 atom of a nucleotide in the opposite strand Two such ribose pairs make up the known ribose zippers however longer zippers comprising more pairs could be envisaged to exist in large RNA folds Assembling modules into RNA architectures packing and connecting helices Doublestranded helices pack together in space to give rise to the intricate threedimensional archi tectures observed in RNA folds Strobel 8 Doudna 1997 The helix modules are connected by distinctly structured single strands and tertiary contacts The connecting regions themselves can form RNA structural motifs such as turns loops 1 and i p 1 pt 1 1 which are found conserved in different RNA architec tures Filling space with cylinders stacking and docking of RNA helices Close packing of doublestranded helices which govems the architecture of RNA folds relies on two principles of assembling cylinders namely endtoend stacking and sidebyside docking The preference of RNA helices for endtoend stacking has been known for a long time as it dominates the arrangement of duplex fragments into long quasi continuous helices in crystals Holbrook 8 Kiln 1997 Likewise helix stacking dominates the shape of RNA folds by defining the length of the principal helical modules tRNA comprises four helices which by pairwise stacking assemble into the Lshaped tRNA fold Robertus et nl 1974 Kim et ul 1974 Extensive stacking of helices occurs in the group I ribozymes illustrated by crystal struc tures which reveal two pairs of coaxially stacked helices in the P4 P6 domain Cate et al 1996a and no less than four such endtoend stacked helices in the P3P9 domain Golden et al 1998 In addition to tRNA and group I ribozymes all other RNA folds for which the threedimensional struc tures have been solved to date contain coaxial stacks of helices namely one in the hammerhead ribozyme Pley et nl 1994a Scott et nl 1995 and two each in the HDV ribozyme Ferr D Amar et nl 1998 and the L11binding domain of 23 S rRNA Conn et al 1999 Wimberly et nl 1999 Whereas helix stacking mediated by the inher ently at surface of terminal basepairs leaves little variety in the configuration of the resulting assem bly docking of helices via their sides comes in many ways And so do the molecular handles that mediate helix docking in RNA architectures In the P4 P6 domain of group I ribozymes two helical stacks are docked sidebyside locked in place by a number of RNA interaction motifs which are grafted into the helices Cate et al 1996a Interactions between the unperturbed heli cal parts involve 2 OH groups of the RNA back bone Such 2 OHmediated contacts of helix sides have been seen in the crystal packing of double stranded RNA helices Holbrook et nl 1991 Schindelin et al 1995 Baeyens et al 1995 Lietzke et nl 1996 Roughly parallel packing of helices occurs in the threedimensional structures of the HDV ribozyme Ferr D Amar et nl 1998 and the L11binding domain of 23 S rRNA Conn et al 1999 Wimberly et nl 1999 with extensive intermingling of helices and connecting re ions In the HDV ribozyme compact packing is additionally enforced through formation of a pair of nested pseudoknots see below Ferr D Amar et al 1998 The compact ness of the HDV ribozyme is attested by the strong cohesion forces within the core of this RNA fold which allow for its catalytic activity even under highly denaturing conditions reviewed by Been 8 Wickham 1997 Stitching Together RNA Tertiary Architectures Singlestranded connections bulges turns and loops The focus of the description of RNA structure has traditionally been the formation of basepairs drawing the line between basepaired double stranded and unpaired singlestranded regions In RNA architectures unpaired regions can be as small as a single bulged nucleotide within a helix or as large as a long singlestranded stretch of nucleotides connecting distant helix ends A wide variety of conformations of nucleotides in single stranded regions is known re ecting the diversity of threedimensional structures of such loops and junctions In some cases however the confor mational pattern of adjacent nucleotides are con served characterizing distinct classes of motifs such as tetraloops and tums Figure 4 Bulgedout residues can be found in helices and given a wider definition in loops and other com pact RNA motifs Single unpaired bases within an RNA doublestrand are depending on the structur al context either stacked into the helix Figure 4a Borer et ul 1995 Varani et ul 1999 or loopedout with uninterrupted stacking of the anking base pairs Figure 4b Portmann et ul 1996 Intermo lecular interactions affect the conformation of bulges as has been observed for an unpaired ade nosine which is looped out into a protein pocket in the complex between bacteriophage MS2 coat pro tein and a bulgecontainjn hair in Valegard et ul 1994 Convery et ul 1998 but intercalated in the free RNA Borer et ul 1995 Loopingout of single residues is frequently found in hairpin and internal loops which are zip pered up into compact structures by stacking and formation of mismatch pairs see above Such bulges in a loop play key structural roles by introducing exible hinges in the RNA backbone providing recognition sites for proteins and other domains in large RNAs and acting as aps close over substrate binding cavities Depending on their structural function ippedout residues are conserved when the base is involved in specific interactions or they are nonconserved when the bulge serves as a mere source of back bone exibility In n FMN aptamer RNA a nonconserved bulgedout base within a mismatchpaired intemal loop allows the formation of an adjacent base triple functioning as a stacking platform for the FMN substrate Fan et ul 1996 A uridine residue ipped into the shallow groove increases back bone exibility contributing to the widening of the deep groove where Tat protein binds to BIV TAR RNA Puglisi et ul 1995 Ye et ul 1995 A con served single bulged adenosine base within a hair pin loop has been discussed as an interaction site for proteins or RNA tertiary contacts in spliced leader RNA Greenbaum et ul 1996 In the L11binding domain of 23 S rRNA conserved bulgedout residues introduce substantial distor tions into regular RNA helices allowing intimate packing and tertiary interactions Conn et ul 1999 Wimberly et ul 1999 Larger RNA bulges with several ippedout resi dues are seldom unstructured but display different mechanisms of conformational stabilisation such as intermolecular basepairing stacking and metal ion binding In the bulgehelixbulge splice site RNA Diener 8 Moore 1998 and the leaddependent ribozyme Wedekind et ul 1999 threenucleotide bulges are stabilized by extrahelical base stacking The three ippedout residues in the leaddepen dent ribozyme form intermolecular basepairs in the crystal packing and are involved in metal ion binding The eminent role metal ions play in bulge stabilization illustrated by extensive cation bind ing in the UCU bulge of HIV1 TAR RNA Ippolito 8 Steitz 1998 and the Arich bulge of the P4 P6 domain of group I ribozymes Cate et al 1996a will be discussed below The reduced conformational restrictions in singlestranded regions of RNA allow for reversing the direction of the backbone Several structural families of such turns are known classified by the nucleotide preceding the turning phosphate U and C turns or by the appearance of the backbone Stum Figure 4ce Utums are stabilized by specific hydrogen bonding and basephosphate stacking interactions across the bend Figure 4c Turn stabilization involves however only few functional groups of the bases leaving free many specific sites for tertiary interactions Uturns following a UNR consensus N is any nucleotide R is a purine were first identified in the anticodon loop and Tloop in tRNA Quigley 8 Rich 1976 Later they have been found in other RNAs such as the hammerhead ribozyme Pley et al 1994a Scott et ul 1995 a hairpin loop Huang et ul 1996 and pseudoknots Kolk et ul 1998 The L11binding domain of 23 S rRNA con tains no less than three Utums along with one S tum see below within a stretch of 58 nucleo tides indicative for the compactness of this RNA fold Conn et ul 1999 Wimberly et ul 1999 It has recently been pointed out that certain tetraloops see below form a distinct class of Utums despite the tuming phosphate belonging to a G in these loops Iucker 8 Pardi 1995 Uturnlike confor mations have also been observed for the loop regions folding around the antibiotic substrate in the solution structures of different aminoglycoside RNA aptamers Iiang et ul 1997 1999 Iiang 8 Patel 1998 In the crystal structure of a ribosomal frameshifting pseudoknot a Cturn has been described which shares some structural features with the Utum Su et ul 1999 The term reversed Uturn has been coined for a motif found in an isolated RNA hairpin of the HDV ribozyme Kolk et ul 1997 In the reversed Ut um the U base directly follows the turning phosphate instead of preceding it and the stacking between bases and phosphate groups is changed Figure 4d 836 Stitching Together RNA Tertiary Architectures a Figure 4 Unpaired regions in RNA comprise single residues termed bulges and whole stretches ot several cone gt gt gt 11013110 secutive bases not involved in either WatsorkCan or stacked i id 1 found to an loo ns 6 an RNA he s 1995 1 Alternatively unpaired residues c a in the stal structure ot an Arform d luch the succe w uri base Kolk at Ll 1997 e In Saturn inverted pucker cztendo w ch introduces an Sasha e RRE R A Battiste at Ll 1995 i an end ot a duplex Arnon t tra oo and UNCG as in the g UUCG P1 hairpin ot grou stab ty due to basepairing between the tu as well as in c d Urtums several ot the stacking bases in RNA domains In contrast to Uturns which globally reverse the RNA strand direction Sturns introd e a locally reversed backbone maintaining an overall linear conformation of the RNA Figure 4e A ipped sugar moiety pointing with its 5 end towards the 3 direction of the RNA strand is central to Sturns und in several ribosom fragments Szewczak at LIL 1993 Szewczak t3 Moore 1995 Wimberly at LIL 1993 1999 Correll r an adenosine p out without in rr uplex containing an a en tm39 e active site ot the hammerh phosphat s a locally reversed backbon ink in th RNA nical baserpail39s a Single unpaired bases can be in the stem ot a 24 nt hairpin loop Borer at Ll te upting the stacking ot the anking aserpairs ul e ortmann at Ll 1995 cre Singlet n tormation causing abrupt changesi b ckbone direce ead ribozyme Pley at id 19943 base which stacks on the d osirteb e is inverted both preceding the sta e is caused by a e Stran a zymes Allain amp v h 39 st and last residue and extensive stacking interactions in i GNRA loops 39 the turn 39 the are available tor tertiary contacts with o r at LIL 1998 Conn at LIL 1999 HIV Revresponse element RRE Battiste at LIL 1996 a Revbinding aptamer RNA Ye at LIL 1996 and a theophylljne aptamer Zimmermann at LI 1997 Singlestranded regions connecting the two strands of a duplex stem are termed hairpin loops constituting c on building blocks of RNA architectures Phylogenetic analyses have revealed that hairpin loops comprising four residues tetra Stitching Together HNA Tertiary Architectures loops are the most frequent loops in large RNAs such as selfsplicing introns rRNA RNaseP and viral genomes Tuerk et ul 1988 James et ul 1988 Woese et ul 1990 Michel 8 Westhof 1990 The evolutionary success of tetraloops has been attribu ted to their conformational stability and their abil ity to participate in tertiary contacts with other RNA motifs Michel 8 Westhof 1990 1996 Abramovitz 8 Pyle 1997 The most extensively studied GNRA N is any nucleotide R is A or G and UNCG tetraloops achieve their stability through the formation of closing sheared G A and G U pairs stacking interactions of the two remain ing unpaired residues and a network of hydrogen bonds Figure 4f and g Cheong et ul 1990 Heus 8 Pardi 1991 Iucker et ul 1996 The extra ordinary rigidity of GNRA tetraloops is attested by the finding that the conformation of a GAAA loop in an RNA hairpin observed by NMR Heus 8 Pardi 1991 is virtually identical with that found in the crystal structure of the P4P6 domain of group I ribozymes Cate et al 1996a Larger loops may adopt the favourable GNRA conformation by excluding the additional nucleotides from the loop scaffold The extrusion of one base has been seen in the pentanucleotide loop of the boxB RNA hair pin which forms a GNRAlike structure in the com plexes between the RNA and phage N proteins Cai et ul 1998 Legault et al 1998b The excluded base is available for interactions with proteins In larger hairpins a considerable content of pur ines often allows extensive mismatch base inter actions leaving only few unpaired residues Examples are provided by the sarcinricin loop of 28S rRNA Szewczak et ul 1993 Szewczak 8 Moore 1995 and the spliced leader RNA hairpin in which only three of 11 singlestranded loop resi dues are pointing out in the solvent while eight bases are involved in stacked mismatch pairs Greenbaum et ul 1996 Unpaired residues may be nonconserved such as the second nucleotide in GNRA tetraloops indi cating a role of the sugar backbone rather than of the base for the loop structure In other hairpin loop fragments however unpaired residues with out base contacts inside the loop are conserved pointing to a participation of the loop in either a tertiary contact or protein binding This is seen in the solution structure of the isolated P loop of 23 S rRNA which is closed by a noncanonical G C pair and contains three highly conserved splayedout guanosine bases which in the full 23 S rRNA are involved in tertiary contacts Viani Puglisi et ul 1997 Pseudoknots A pseudoknot may be considered as consisting of two fused hairpins in which the loops mutually provide bases for the formation of two stems Clas sically pseudoknots are described as elements of RNA tertiary structure derived from basepairing between a hairpin loop and a complementary singlestranded sequence close to the hairpin Pleij et ul 1985 Puglisi et ul 1988 The two basepaired stems are stacked upon each other at the junction Hilbers et ul 1998 giving rise to an extended architecture Figure 5a Helix arrangement in pseudoknots is subject to some conformational exibility as both linear and bent geometries of the stems have been found in different pseudoknot structures along with either coaxial or noncoaxial stacking In noncoaxially stacked pseudoknots considerable rotation between the adjacent stems along with shifting and tilting of the helix axes occurs giving rise to an overall kinked geometry as has been observed in a ribosomal frameshifting pseudoknot Su et ul 1999 In some pseudoknots the two helices are separated by intervening nucleotides which can stack between the stems introducing a bent junction Chen et ul 1996 The kinked shape plays an important role in the bio logical role of frameshifting pseudoknots Chen et ul 1996 Su et ul 1999 The connecting loops in pseudoknots cross the grooves of the helices so as to position loop 1 in the deep groove of stem 2 and loop 2 in the shal low groove of stem 1 In order to accommodate the single strands the geometry of the stems may deviate significantly from standard Aform RNA helices Su et ul 1999 The singlestranded loops can thus participate in tertiary contacts in the RNA grooves attested by a triplestrand inter action of a sin le base discovered in a classical pseudoknot Kolk et ul 1998 and triplex formation over six layers in a ribosomal frameshifting viral pseudoknot see above Su et ul 1999 Distortions of the Aconformation in pseudoknot helices can be also induced by connecting loops which are too short to cross the grooves of regular stems Pleij et ul 1985 In the crystal structure of a ribosomal r r h q 100 a t is accommodated by overtwisting of the first basepair in the adjacent stem Su et ul 1999 The tight packing of stems and singlestranded regions in pseudoknots leads to highly compact folds which are stabilized by metal ion binding see below and extensive tertiary hydrogen bond ing giving rise to characteristic RNA structural motifs such as base quadruples Su et ul 1999 and ribose zippers Ferr D Amar et ul 1998 see above Two nested pseudonots are responsible for the extraordinary stability of the HDV ribozyme the threedimensional fold of which beautifully demonstrates RNA s capacity to form intricate compact architectures Figure 5b Ferr D Amare et ul 1998 Interactions of loops kissing and docking Loops are stabilized by interactions often invol ving only few functional groups of the bases which are thus available for tertiary contacts with other RNA domains Such tertiary interactions comprise looptoloop kissing complexes and looprecep tor complexes in which a loop docks into a double 838 Stitching Together RNA Tertiary Architectures d nusmatchpairing and hydrogen bonding to the backbone t An interrn Figure 5 Compact folding of RNA architectures depends on cork served tertiary interaction mo39s s a b seudoknots and cm longrrange contacts involving orm tertiary interactions with other RNA motifs such as cork served tetraloop 6 receptors and t helices e In the Pips domain o 510 1 ribozyrnes a to a receptor module 1995a which provides sf olecular contact in crystals of a 3 E E on ribozyrne provides an example for a GAAA tetraloop interacting with the shallow groove of a hehx Pley at Ll 1994b stranded receptor motif An interaction between two loops has already been found in tRNA namely between the D and Tloops which how ever are stabilized b of e 39 Kissing complexes are formed b between the singlestran ed residues of sequence complementary loops To date structural data are available for two RNA kissing contacts namely of the CDZEI inverted loop RNA arino at LIL 1995 Lee amp Crothers 1998 and the HIV2 TAR hairpin Chang amp Tinoco 1997 in complex with their respective complementary loops Figure 5cd These complexes represent transient structures which 111 viz0 precede the formation of extended sense antisense RNA duplexes The kissing com exes are however stable RNA architectures which are specifically recognized by proteins Eguchi amp Tomizawa 1990 In both the TAR and CDZEI kissing complexes all loop residues partici ate in intermolecular base p 39 39 creating double helices of respectively six and seven WatsonCrick pairs The duplex formed by the kissing loops stacks on both sides coaxially with the anking hairpin helices The overall struc ture of the kissing complexes resembles a bent quasicontinuous helix The extensive basepairing between the loops forces several of the backbone phosphate groups into close proximity giving rise to quotphosphate clusters Lee amp Crothers 1998 which are stabilized by metal cations see below Iossinet at LIL 1999 tetr oops see above are frequently found in large RNAs such as rRNA selfsplicing 39 trons and RNase P with these motifs playing crucial roles in proper folding by providing long ran e tertia 39 actions with a ro riate dock ing sites Costa amp Michel 1995 Such contacts first suggested based on phylogenetic analyses and modeling studies Michel amp Westhof 1990 Iaeger at LIL 1994 have later been confirmed by crystal 39 e in which GAAA tetraloops dock into either a helix or a tetraloop receptor In crystal ack39 of a hammerhead ribo zyme intermolecular contacts are formed between the three adenosine bases of a GAAA tetraloop in Stitching Together RNA Tertiary Architectures one RNA molecule and the shallow groove edges of consecutive GC basepairs in a second molecule Figure 5f Pley et al 1994b In addition to hydrogen bonds between the bases interactions involve 2 OH groups of the residues in both the tetraloop and the helix A similar contact between a tetraloop and the shallow groove of an RNA duplex has been identified for the L9 GAAA loop docking into the P5 stem in the group I ribozymes Golden et ul 1998 Also in group I ribozymes but in the P4 P6 domain a tertiary interaction has been observed between a GAAA tetraloop and a specific RNA receptor module Figure 5e Cate et al 1996a The tetraloop receptor motif comprises 11 con served nucleotides Costa 8 Michel 1995 five of which are located in an internal loop zippering up into a compact duplexlike structure with an opened shallow groove and nearly coaxial align ment of the anking helices The key feature of the tetraloop receptor is a notch resulting from a reduced backbone rise due to base pairing between two consecutive adenosine bases in an adenosine platform see above The platform opens up the shallow groove and serves as a stacking surface for the pivotal adenine of the GAAA tetraloop which docks into the notch within the receptor motif As in the tetraloophelix contact hydrogen bonds involvin both bases and 2 OH roups of the backbone contribute to the stability of the tetra loopreceptor interaction motif The tetraloop receptor itself is stabilized by a potassium ion specifically bound within a pocket below the ade nosine platform see below Basu et ul 1998 A peculiar example of an internal loop docking into a receptor structure is provided by the hairpin ribozyme This catalytic RNA motif consists essen nucleotides zippers up by mismatch formation into an 0 en du lex with an extremel widened shallow groove Figure 2c Butcher et ul 1999 which serves as a docking surface for loop A Cai 8 Tinoco 1996 Eamshaw et ul 1997 Adding glue to RNA folds metal ions and water RNA is a polyanion which binds cations and water in order to acquire its biologically functional threedimensional structure In e absence of metal ions all known complex RNA folds form most of their secondary structure but little if any tertiary structure In many crystal structures of RNA molecules metal ions have been found at specific binding sites reviewed by Feig 8 Uhlenbeck 1999 Some catalytic RNAs termed ribozymes use metal ions in their active sites reviewed by Pyle 1993 Carola 8 Eckstein 1999 McKay 8 Wedekind 1999 Evidence for stable conserved water molecules participatin in RNA structures comes from highresolution crystallo graphic analyses Both metal ions and water can thus be considered as intrinsic parts of RNA three dimensional architecture W esthof 1988 Di and monovaent cations At physiological pH RNA molecules are associ ated with a number of counter ions corresponding to that of the negatively charged nucleotides While the majority of cations are delocalized con tributing to nonspecific counter ion condensation some ions occupy specific ion binding sites pre cisely defined by the threedimensional folding of the RNA chain Laing et ul 1994 These metal ion binding sites Figure 6 are positioned within local mininia in the electrostatic field created by the charges on the RNA atoms Hermann 8 Westhof 1998a The important role of metal ions for RNA threedilnensional folding was recognized early when it was found that cations are necessary for the stabilization of the native structure of tRNA Fresco et ul 1966 Crystal structure analysis has later revealed the positions of four Mg bound to RNA E in loop regions Figure 6a and b Holbrook et ul 1977 Jack et ul 1977 Quigley et ul 1978 Specific binding of metal ions to RNA can occur either directly through innersphere coordination Figure 6a or can be mediated by the hydration shell via outersphere contacts Figure 6b The interaction between RNA and directly bound metal ions is governed by a competition between electro static and hydration forces both of which are decreasing functions of ionic radius and increasing with ionic charge Draper 8 Misra 1998 Small divalent ions such as Mg bind in innersphere coordination alInost exclusively to the negatively charged phosphate groups of RNA while inter actions with the hydration shell of Mg also involve other less electronegative sites such as purine N7 and carbonyl oxygen atoms of the bases Metal ions stabilize the close approach of phos phate groups at the interface between RNA strands as has frequently been observed in crystal packing Holbrook 8 Kiln 1997 and in strand junctions associated with complex folds such as the P4P6 domain of group I ribozymes Cate et al 1996a and the L11binding domain of 23 S rRNA Wimberly et ul 1999 Speci c pockets for the binding of divalent metal ions are formed in loops and bulges where the RNA backbone folds back on itself bringing phosphate groups in close proxi mity Such metalstabilized folded backbone struc tures have been found in the tum connecting the acceptor stem and the D loop in tRNA Figure 6b Holbrook et ul 1977 Jack et ul 1977 Quigley et ul 1978 in the Arich bulge of the P4 P6 domain of group I ribozymes Figure 6c Cate et al 1996a in the UCU bulge in HIV TAR Figure 6d Ippolito 8 Steitz 1998 in the L11 inding domain of 23 S rRNA Wimberly et ul 1999 and in kissin loop complexes Lee 8 Crothers 1998 Iossinet et ul 1999 840 Stitching Together RNA Tertiary Architectures a g the deep groove edge ot a sheared GA pair in an RNA duplex Baeyens at Ll 1995 i bound to the dee i by severa o light blue Water ligands are shown as red spheres in a The requirement of certain bulges for metal ions sence of high ion concentrations are available In HIV1 TAR RNA the UCU bulge is exible in low salt solution Aboulela at LIL 1996 but forms a rigid metal ionstabilized structure in stal in the presence o 39 2 Figure 6d Ippolito amp Steitz 1998 In the crystal tructur o ead ndent ribozyme the backbone of the GAG bulge is tethered to the deep gro h drated M w 39ch interacts with two phosphate groups of the bulge and two guanosine bases in the adjacent stem Figure 6e i Speci c binding sites tor mono osonnal ameshittin d 1 see also Figure 5a al ions as spheres colored according to arc e g and arnrn b and 1 water ligands ot the hydration shell ot cations have been omitted tor Figure 6 Metal ions are an inte gral art ot RNA archi e where the p ot tRNAP Iolbrook at Ll 1977 gley t a Mgztbinding site at a EU tandenn pair in e 7P onnain ot group I ribozyrnes Cate at al 1995a and divalent cation coordinated at h A stack ot groove in the lu hrsalt crystal structure ot th pair ot binding sites which due to their cl t g seu oknot Su at Ll 1999 Phosphate groups are s own as grey stick h Mg violet on Ca cyan 0 Osam yellow i Nat ine in green in t In all panels except tor a clarity tymg e tetrahedra Wedekind S McKay 1999 whereas in lowsalt solution the bases of the bulge are pointing inside the groove oogstraten at LIL 1998 In the Arich bulge of group I backbone phosphate ribozymes the toll ws a rk crew turn positioning groups together with Mg in a cluster lnslde the loop while the bases are oriented towards the outside Cate at LIL 1996a The metal t1on of nu RNA bases oriented to the core and 39 39 side preV r aniza modules with their their backbone lining the surface is turned ut Amon he nu metal i P6 domain of group I ribozymes it was primarily Stitching Together RNA Tertiary Architectures the Arich bulge which led to the proposal that RNA folds around a metal ion core Cate et ul 1997 in analogy to the hydrophobic core of proteins In addition to the formally charged phosphate groups of the backbone electronegative atoms of the bases contribute to specific metal ion binding in RNA architectures This becomes evident especially for deep groove regions where the elec trostatic field of regular helical RNA is perturbed by noncanonical basepairs A minmal change of duplex geometry which is introduced by a G U wobble pair followed by a Y G pair is sufficient to create specific cationbinding pockets in the deep groove Cate Doudna 1996 Hermann 8 Westhof 1998a The uridine O4 atom which is not involved in hydrogen bonding along with the N7 and 06 atoms of guanosine contribute to cat ion coordination at the G U air Metal ion bind ing sites at the 5 GU3 3 YG 5 motif have been found in the anticodon stemloop of tRNAAsp Westhof et ul 1985 and in the P4P6 domain of group I ribozymes Figure 6f Cate et al 1996a Cate 8 Doudna 1996 Kieft 8 Tinoco 1997 Colmenarejo 8 Tinoco 1999 The 5 GU3 3 YG5 motif occurs frequently in large RNAs such as rRNA Gautheret et ul 1995 and might be of general mportance as a metal ion binding site in RNA folds Sheared G A pairs see above are another example for noncanonical basepairs associated with cation binding sites Divalent metal ions have been found in an RNA duplex Figure 6g Baeyens et ul 1996 and in hammerhead ribo zymes Pley et al 1994a Scott et ul 1995 at the deep groove edge of guanosine in WatsonCrick CG pairs which are followed by a sheared G A pair At the 5 CG3 3 GA5 motif cations bind to the N7 atom of the CG and the phosphate group of the G A which due to the backbone con formation introduced at the G A pair are posi tioned to line an electronegative pocket Mg binding at a sheared G A pair has also been found in the loop E of 5 S rRNA in which a stack of three noncanonical basepairs centered at the G A is stabilized by extensive metal ion bindin in the dee roove Figure 6h Correll et ul 1997 This metal ion zipper which forces a sig nificant narrowing of the deep groove might par tially form due to the crystallization conditions at high Mg concentrations While a considerable number of nucleic acid threedimensional structures containing divalent metal ions are available examples for monovalent cations specifically bound to either DNA or RNA are scarce with the exception of G quadruplexes which are stabilized by alkali metal ions between the layers of the G tetrad planes Kang et ul 1992 Cheong 8 Moore 1992 Laughlan et ul 1994 Hud et ul 1996 Kettani et ul 1998 The role of mono valent metal ions as a part of RNA threedmen sional structure has been acknowledged only recently Wang et ul 1993 Draper 8 Misra 1998 despite it being known for some tune that sodium ions can subsitute for Mg in the correct folding of tRNA Crothers 8 Cole 1978 The growing mogenetic approaches see below for identifying the binding partners of individual RNA chemical groups reviewed by Strobel 1999 have permitted the discovery of specific pockets for monovalent metal ions in RNA architectures In the crystal structure of a ribosomal frameshift ing pseudoknot a sodium ion is located at a key position tethering the singlestranded loop 2 into the shallow groove of stem 1 Figures 5a and 6i Su et ul 1999 The sodium ion binds to a phos phate group of the loop and is positioned in the plane between two purines coordinating through nitrogen atoms to the ion The tight packing of loops and stems requires the presence of metal cat ions for the stable folding of pseudoknots Wyatt et ul 1990 Kolk et ul 1998 which contain binding sites for divalent metal ions Hermann 8 Westhof 1998a Gonzalez 8 Tinoco 1999 A high concen tration of monovalent cations can however substi tute for divalent metals in pseudoknot folding Wyatt 8 Tinoco 1993 Adenosine platforms conserved building blocks of RNA architectures see above contain a specific binding site for monovalent metal ions as an inte gral part of their structure Basu et ul 1998 In the P4P6 domain of group I ribozymes monovalent ions have been found immediately below adeno sine platforms Cate et al 1996b Basu et ul 1998 stabilizing the intrastrand sidebyside AA pair by coordinating to the phosphate group between the adenosine bases and to bases of the noncanonical pair which stacks below Since adenosine platforms are a key structural element of the tetraloop recep tor module sitespecific binding of monovalent cations is mportant for the formation of correct tertiary structure in a wide variety of large RNAs which contain the tetraloopreceptor contact In addition to the role they play in RNA three dimensional folding metal ions are constituents of the active site of selfcleaving catalytic RNAs such as the selfsplicing group I and II introns Streicher et ul 1996 Deme et ul 1999 Sontheimer et ul 1999 RNaseP Smith 8 Pace 1993 hammerhead ribozyme Dahm 8 Uhlenbeck 1991 Scott 8 Uhlenbeck 1999 and the leaddependent ribozyme Pan 8 Uhlenbeck 1992 Crystal structure ana lyses have revealed positions of putative catalytic metal ions in the hammerhead and leaddependent ribozymes Wedekind 8 McKay 1999 Pley et al 1994a Scott et ul 1995 1996 In the active site of a group I intron two metal ions have been posi tioned in a threedimensional model of the catalytic core Streicher et ul 1996 resembling a geometry proposed earlier Steitz 8 Steitz 1993 The divalent cations suggested to participate in catalysis in both the hammerhead and leaddepen dent ribozymes are directly coordinated to the scis sile phosphate group The cations at the active site 842 Stitching Together RNA Tertiary Architectures of the ribozymes drive the chemistry of the clea vage reaction reviewed by Kuimdis amp McLaughlin 1998 by providing a metalbound hydroxide which can deprotonate the 2 OH group for nucleophilic substitution at the adjacent phos phate Based on molecular dynamics simulations it has been suggested that the hydroxide 39 volved in the catalysis of the hammerhead ribozyme Dahm at LIL 1993 is bridging between two Mg in proxi mity of the cleavage site Hermann at LIL 1997 Ordered water in RNA structures Stable hydration patterns in surface depressions pockets and the grooves of duplexes contribute to the stability of RNA architectures Figure 7 Metal binding sites in RNA folds see above inevitably contain restricted water molecules o t e hydration shell around the cations Highresolution Xray c stallo a hy permits the identi cation of the positions of tightly bound water molecules in RNA architectures Organized networks of metal hydration water have been found in crystal struc tures of the loop E of SS rRNA Correll at LIL 1997 HIV1 TAR RNA Ippolito amp Steitz 1998 a ri osomal frameshiftl39ng pseudoknot Su et LIL 1999 and tRNAs Westhof 1988 In tRNAs a regular hydration pattern has been observed around the backbone in doublestranded re 39ons wh re water molecules bridge successive phosphate groups of each strand Westhof at LIL 1988 Similar stable arrangements of water are seen in Aform RNA duplexes in which solvent molecules orm transversal brid es between the strands across the shallow groove and regular longitudinal h dration motifs in the deep groove Figure 7b Egli at LIL 1996 Th r 39 39 of water at phosphate groups is due to both hydrogen bonding o 39 3quot m aro d these sites The effe o restriction of water molecules gives rise to the crys tallographic quotwater sites in the anticodon loop of tRNAs Westhof at LIL 1985 1988 which have been shown to coincide with electronegative mini ma in the electrostch field around the RNA Hermann amp Westhof 1998a Like metal ions water molecules occupy electronegative pockets thereby stabilizing the folding of the RNA chain Extensive association of te 39 ordered solvent has been found in the crystal struc tures of tRNAs Westhof at LIL 1985 1988 Orga nized networks of restricted water molecules are Figure 7 Water molecules forming sta a The interpenetration of RNA threedim demonstrated by the crystal structure of a patterns are associated with hall w groove e examp e a s o dges o U h v p the 6 loop E of 5 s rRNA Correll at Ll 1997 d At the deep groo bilizjng hydrogen ensional struc the backbone of Arform R ctiire of an RNA duplex Egli at Ll 1995 Many mismatch baserpairs are stabilized b olar groups in both bases wi bond networks are an intrinsic part of RNA architectures m e b locally restricted water molecules is y osomal pseudoknot Su at L2 1999 1 Regular hydration NA in w a r ridge successive a e th hydrogen bonds Such w ter brid airs as seen in c tRNA w ve edge a water molecule is ou stab Zing a RNA duplex Baeyens at L22 1995 Water molecules are shown as red spheres and a Na in UvU rmsmat m an a is colored in light blue Stitching Together RNA Tertiary Architectures located at the surface of a Cturn see above and mediate the tertiary interaction of loop 1 in the deep groove of stem 2 in a ribosomal frameshifting pseudoknot Figure 7a Su et ul 1999 Some of the stable hydration patterns discussed above involve the bases in hydrogenbonded pairs which are surrounded by systematic arrangements of water Auffinger 8 Westhof 1998 Single brid ging water molecules form hydrogen bonds between unoccupied polar groups of the bases in many mismatch pairs Correll et ul 1997 Leontis 8 Westhof 1998a In G U pairs the U is retracted towards the deep groove creating a depression on the shallow groove surface which is a conserved site of specific hydration with the occupying water molecule bridging the two bases of the wobble pair Westhof 1988 Such precisely defined water molecules at G U pairs have been found in the crystal structures of tRNAs Figure 7c Westhof et ul 1988 RNA duplexes Mueller et ul 1999 and the loop E of 5 S rRNA Correll et ul 1997 In contrast to G U wobble pairs for which the bridging water molecule is located in the shallow groove U U mismatches are stabilized by a water molecule in the deep groove forming hydrogen bonds between the O4 carbonyl atoms of the uri dine bases Figure 7d Baeyens et ul 1995 Con served water binding sites linking the bases in the shallow roove are also found at G A mismatch pairs Figure 7e Leonard et ul 1994 Correll et ul 1997 In the crystal structure of the loop E of 5 S rRNA three consecutive mismatches centered at a sheared G A are stabilized by water mol ecules alternatingly bridging the shallow and de groove edges of the basepairs Correll et ul 1997 These noncanonical basepairs are connected by single hydrogen bonds and thus require water molecules in order to obtain a stable basepairing scheme In addition to their function for stabilizing RNA threedimensional structure water molecules play an important role in recognition processes In the interactions between RNA architectures and both proteins and small molecules water molecules pro vide plasticity at the intermolecular surfaces insur ing specific recognition and highaffinity binding The shape of things to come some future prospects of understanding RNA architecture and function Analyses of the many threedimensional struc tures of RNA molecules becoming available over the last decade have answered important questions about the principles that govern the assembly of RNA architectures reviewed by Batey et ul 1999 Moore 1999 this review While our understand ing of RNA structurefunction relationships is now deeper beyond the surface once scratched when the first crystal structures of tRNAs had been solved some of the most exciting challenges of the RNA world still await solutions Among these challenges are the threedimen sional structures of large RNAs and RNAprotein complexes especially those of selfsplicing group II introns RNase P and the multicomponent assem blies of the ribosome and the splicing machinery Working models of the RNA component of RNase P Massire et ul 1998 and 16 S rRNA Malhotra 8 Harvey 1994 Mueller 8 Briniacombe 1997 Masquida et ul 1997 are available It will be inter esting to see if these models are in similar good agreement with experimental structures yet to be determined as was the model for the group I ribo zyme Golden et ul 1998 Despite being ephemeral approximations of mol ecular architecture structural models will continue being important amplifiers for our thinking about structurefunction relationships in the RNA world especially with the aim of designing new exper iments The construction of RNA molecular models will greatly benefit from chemogenetic techniques which allow the probing of single chemical groups for their contribution to the function of an RNA fold reviewed by Strobel 1999 The elucidation of the crystal structures of the complete ribosomal subunits has been a challenge of longstanding interest reviewed by Yonath 8 Franceschi 1998 Moore 1998 Significant progress towards these goals has recently been achieved for both the isolated small and large subunits and the complete functional ribosome For the isolated sub units electron density maps of around 5 A resol ution are available Clemons et ul 1999 Ban et ul 1999 The crystal structure of a 70 S ribosome functional complex containing also tRNAu and a piece of mRNA has been refined to 78 A Cate et ul 1999 Since the crystals of the subunits and the functional ribosome diffract to higher resol ution one can anticipate an exponential increase in our understanding of RNARNA and RNAprotein interactions in the forseeable future Static threedimensional architecture alone can provide only partial insights into the function of molecular machines such as the ribosome which require dynamic exibility in order to selfassemble and accomplish their tasks in the cell The import ance of exibility for RNA function is highlighted by the simplest RNA molecular machines namely the ribozymes which catalyze chemical reactions in the absence of proteins In the small autocleaving RNAs such as the hammerhead and leaddepen dent ribozymes the scissile phosphodiester bond is located in a exible region of the backbone Hoogstraten et ul 1998 Legault et al 1998a Wedekind 8 McKay 1999 Scott et ul 1996 Murray et ul 1998 Defined conformational changes of the backbone have been found to be crucial for the cleavage reaction to proceed in these ribozymes reviewed by Lilley 1999 McKay 8 Wedekind 1999 In the hammerhead ribozyme a sugar moiety of the nucleotide preceding the scis sile phosphodiester bond must ip its ring confor mation in order to position the 2 OH nucleophile in the proper orientation for the substitution at the Stitching Together RNA Tertiary Architectures adjacent phosphate group Hermann et al 1997 Murray et al 1998 In a manner similar to the efforts to study the significance of structural rearrangements for RNA function the understanding of the roles base and backbone modi cations play in RNA architectures is still in its infancy While it is known that modi fied nucleotides are required for the precise recog nition of tRNAs by proteins reviewed by Grosjean 8 Benne 1998 molecular insight into the in uence of modifications on RNA structure and exibility are yet scarce In some cases modified nucleosides contribute to the thermostability of RNAs in ther mophilic organisms Edmonds et al 1991 For a basemethylated GGAA loop in 16 S rRNA it has recently been shown that the methyl groups induce a stable yet completely different conformation compared to that of canoncial GNRA tetraloops Rife 8 Moore 1998 It is likely that the peculiar conformation of the methylated loop is crucial for its function in rRNA In the above discussion the importance of RNA architecture for recognition by small molecules and proteins has occasionally been mentioned Indeed the large majority of cellular RNAs function in con cert with partner molecules either as stable perma nent complexes such as the ribosome or in transient associations such as the mRNA splicing machinery The potential specificity of interactions between RNAs and small molecules renders RNA a prime target for therapeutic intervention reviewed by Hermann 8 Westhof 1998b Afshar et al 1999 especially because various RNA mol ecules are involved in all stages of gene expression While the rational design of drugs which specifi cally recognize RNA motifs is still in its infancy promising strategies for developing small mol ecules targeting RNAs and RNAprotein com plexes have been outlined reviewed by Hermann 2000 The growing understanding of RNA archi tecture and its correlation with function will pro vide firm grounds for the development of specific RNAdirected effector molecules First steps have been undertaken to unravel the principles of molecular recognition in complexes between RNA and proteins reviewed by De Guzman et al 1998 Cusack 1999 Draper 8 Reynaldo 1999 Kambach et al 1999 Steitz 1999 peptides reviewed by Frankel 8 Smith 1998 Patel 1999 Puglisi 8 Williamson 1999 and small molecules reviewed by Feigon et al 1996 Patel et al 1997 Hermann 8 Westhof 1998b Despite the wide diversity of molecular compositions and biological functions of the studied systems a lim ited number of common themes associated with intermolecular interactions involving RNA have been uncovered Satisfyingly the understanding of the threedimensional architectures of the partici pating molecules has allowed the drawing of a pre liminary map of some general routes associated w1 stru tu f1 ti 1 It is this map that will permit future navigation in the immensely complex and challenging world of biological processes which involve RNA Acknowledgements Funding was provided by NIH SM54777 References Aboulela F Karn I 8 Varani G 1996 Structure of R RNA in the absence of ligands reveals a novel conformation of the trinucleotide bulge Nucl Acids Res 24 3974 3981 Abramovitz D L 8 Pyle A M 1997 Remarkable morphological variability of a common RNA fold ing motif the GNRA tetraloop receptor interaction Mal Biol 266 493506 Afshar M Prescott C D 8 Varani G 1999 Struc turebased and combinatorial search for new RNA binding drugs Curr Opin Biatechrwl 10 5963 Allain F H 8 Varani G 1995 Structure of the P1 helix from group I selfsplicing introns Mal Biol 250 333353 Auffinger P 8 Westhof E 1998 Hydration of RNA basepairs Biumul Struct Dynam 16 693707 Baeyens K I DeBondt H L 8 Holbrook S R 1995 Structure of an RNA double helix including uracil uracil basepairs in an internal loop Nature Struct Biol 2 5662 Baeyens K I DeBondt H L Pardi A 8 Holbrook S R 1996 A curved RNA helix incorporating an internal loo with GA and AA nonWatsonCrick basepairing Pruc Natl Acad Sci LISA 93 12851 12855 Ban N Nissen P Hansen I Capel M Moore P B 8 Steitz T A 1990 Placement of protein and RNA structures into a 5 Aresolution map of the 50 S ribosomal subunit Nature 400 841847 Basu S Rambo R P StraussSoukup I Cate I H Ferr D Amar A R Strobel S A 8 Doudna I A 1998 A specific monovalent metal ion integral to the AA platform of the RNA tetraloop receptor Nature Struct Biol 5 986 992 Batey R T Rambo R P 8 Doudna I A 1999 Ter tiary motifs in RNA structure and folding Angew Chem Int Ed Engl 38 2326 2343 Battiste I L Mao H Rao N S Tan R Muhandiram D R Kay L E Frankel A D 8 Williamson I R 19 0c helixRNA major groove recogntion in an HIV1 rev peptide RRE RNA complex Science 273 15471551 Been M D 8 Wickham G S 1997 Selfcleaving ribo Zymes of hepatitis delta virus RNA Eur Biochem 247 741753 Borer P N Lin Y Wang S Roggenbuck M W Gott I M Uhlenbeck O C 8 Pelczer I 1995 Proton NMR and structural features of a Z4nucleo tide RNA hairpin Biochemistry 34 6488 6503 Brion P 8 Westhof E 1997 Hierarchy and dynamics of RNA folding Annu Rev Biaphys Biumul Struct 26113 137 Brodsky A S 8 Williamson I R 1997 Solution struc ture of the HIV2 TARarginineamide complex Mal Biol 267 624639 Brodsky A S Erlacher H A 8 Williamson I R 1998 NMR evidence for a base triple in the HIV2 Stitching Together RNA Tertiary Architectures 845 TAR CGC mutantargininamide complex Nucl Acids Res 26 19911995 Butcher S E Dieckmann T 8 Feigon I 1997 Sol ution structure of a GAAA tetraloop receptor RNA EMBO It 16 74907499 Butcher S E Allain F HT 8 Feigon I 1999 Sol ution structure of the loo B domain from the hair pin ribozyme Nature Struct Biol 6 212216 Cai Z 8 Tinoco L Ir 1996 Solution structure of loop A from the hairpin ribozyme from tobacco ringspot virus satellite Biochemistry 35 6026 6036 Cai Z Gorin A Frederick R Ye X Hu W Majumdar A Kettani A 8 Patel D I 1998 Sol ution structure of P22 transcriptional antitermina tion N peptidebox B RNA complex Nature Struct Biol 5 203 212 Carola C 8 Eckstein F 1999 Nucleic acid enzymes Curr Opin Struct Biol 3 274283 Cate I H 8 Doudna I A 1996 Metalbinding sites in the major groove of a large ribozyme domain Structure 4 12211229 Cate I H Gooding A R Podell E Zhou K Golden B L Kundrot C E Cech T E 8 Doudna I A 1996a Crystal structure of a group I ribozyme domain principles of RNA packing Science 273 16781685 Cate I H Gooding A R Podell E Zhou K Golden B L Szewczak A A Kundrot C E Cech T E 8 Doudna I A 1996b RNA tertiary structure mediation by adenosine platforms Science 273 16961699 Cate I H Hanna R L 8 Doudna I A 1997 A mag nesium ion core at the heart of a ribozyme domain Nature Struct Biol 4 553 558 Cate I H Yusupov M M Yusu ova G 2 Earnest T N 8 Noller H F 1999 Xray crystal structure of 70 S ribosome functional complexes Science 285 20952135 Chang K Y 8 Tinoco L Ir 1997 The structure of an RNA kissing hairpin complex of the HIV TAR hairpin loop and its complement It Mol Biol 269 5266 Chen X Kang H Shen L X Chamorro M Varmus H E 8 Tinoco L Ir 1996 A characteristic bent conformation of RNA pseudoknots promotes 71 frameshifting during translation of retroviral RNA It Mol Biol 260 479483 Cheong C 8 Moore P B 1992 Solution structure of an unusually stable RNA tetraplex containing G and Uquartet structures Biochemistry 31 8406 4 841 Cheong C Varani G 8 Tinoco L Ir 1990 Solution struc re of an unusually stable RNA hairpin 5GGACUUCGGUCC Nature 346 680682 Clemons W M Ma I L C Wimberly B T McCutcheon I P Capel M S 8 Ramakrishnan V 1999 Structure of a bacterial 30S ribosomal sub unit at 55 A resolution Nature 400 833 840 Colmenarejo G 8 Tinoco L Ir 1999 Structure and thermodynamics of metal binding in the P5 helix of a group I intron ribozyme It Mol Biol 290 119135 Conn G L Draper D E Lattmann E E 8 Gittis A G 1999 Crystal structure of a conserved ribo somal proteinRNA complex Science 284 1171 1174 Convery M A Rowsell S Stonehouse N L Ellington A D Hirao L Murra I B Peabody D S Phillips S E V 8 Stockley P G 1998 Crystal structure of an RNA aptamerprotein com plex at 28 A resolution Nature Struct Biol 5 133 139 Correll C C Freeborn B Moore P B 8 Steitz T A 1997 Metals motifs and recognition in the crystal structure of a 5 S rRNA domain Cell 91 705712 Correll C C Munishkin A Chan YL Ren Z Wool L G 8 Steitz T A 1998 Crystal structure of the ribosomal RNA domain essential for binding elongation factors Proc Natl Acad Sci LISA 95 1345613441 Costa M 8 Michel F 1995 Frequent use of the same tertiary motif by selffolding RNAs EMBO It 14 1276 1285 Crothers D M 8 Cole P E 1978 Conformational changes in tRNA In Transfer RNA Altman S ed pp 196 247 MIT Press Cambridge MA Cusack S 1999 RNAprotein complexes Curr Opin Struct Biol 9 66 73 Dahm S C 8 Uhlenbeck O C 1991 Role of divalent metal ions in the hammerhead RNA cleavage reac tion Biochemistry 30 9464 9469 Dahm S C Derrick W B 8 Uhlenbeck O C 1993 Evidence for the role of solvated metal hypdroxide in the hammerhead cleavage mechanism Biochemis my 32 1304013045 Dallas A 8 Moore P B 1997 The loop Eloop D region of Escherichia coli 5 S rRNA the solution structure reveals an unusual loop that may be important for binding ribosomal proteins Structure 5 16391653 De Guzman R N Turner R B 8 Summers M F 1998 ProteinRNA recognition Biopolymers Nucl Acids Sci 48 181195 De los Santos C Rosen M 8 Patel D I 1989 NMR studies of DNA RnY7nYn triple helices in solution imino and 39 n markers of TAT and CGC base triple formation Biochem istry 28 72827289 Deme E Nolte A 8 Iacquier A 1999 Unexpected metal ion requirements specific for catalysis of the branching reaction in a group II intron Biochemistry 38 31573167 Diener I L 8 Moore P B 1998 Solution structure of a substrate for the archaeal pre tRNA splicing endo nucleases the bulgehelixbulge motif Mol Cell 1 883 894 Draper D E 8 Misra V K 1998 RNA shows its metal Nature Struct Biol 5 927930 Draper D E 8 Reynaldo L P 1999 RNA binding strategies of ribosomal proteins Nucl Acids Res 27 381388 Earnshaw D L Masquida B Muller S Sigurdsson S T Eckstein F Westhof E 8 Gait M I 1997 Interdomain crosslinking and molecular modelling of the hairpin ribozyme It Mol Biol 274 197212 Edmonds C G Crain P F Gupta R Hashizume T Hocart C H Kowalak I A Pomerantz S C Stetter K O 8 McCloskey I A 1991 Posttran scriptional modification of tRNA in thermophilic archaea Archaebacteria It Bacteriol 173 31383148 Egli M Portmann S 8 Usman N 1996 RNA hydration a detailed look Biochemistry 35 8489 8494 Eguchi Y 8 Tomizawa I 1990 Complex formation by complementary RNA stemloops and its stabiliz ation by a protein function of ColEl Rom protein Cell 60 199209 846 Stitching Together RNA Tertiary Architectures Famulok M 1999 Oligonucleotide aptamers that rec gnize small molecules Curr Opin Struct Biol 9 324 329 Fan P Suri A K Fiala R Live D 8 Patel D I 1996 Molecular recognition in the FMNRNA aptamer complex It Mol Biol 258 480500 Gesteland R F Cech T R 8 Atkins I F eds 2nd edit pp 287319 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Feigon Dieckmann T 8 Smith F W 1996 Apta mer structures from A to Q Chem Biol 3 611617 Ferr D Amare A R 8 Doudna I A 1999 RNA folds insights from recent crystal structures Annu Rev Biophysl Biomol Structl 28 5773 Ferr D Amare A R Zhou K 8 Doudna I A 1998 Crystal structure of a hepatitis delta virus ribozyme Nature 395 567574 Frankel A D 8 Smith C A 1998 Induced folding in RNA rotein recognition more than a simple hand shake Cell 92 149151 Fresco I R Adams A Ascione R Henley D 8 Lindahl T 1966 Tertiary structure in transfer ribonucleic acids Cold Spring Harbor Symp Quant Biol 31 527537 Gautheret D 8 Gutell R R 1997 Inferring the conformation of RNA basepairs and triples from patterns of sequence variation Nucl Acids Res 25 15591564 Gautheret D Konings D 8 Gutell R R 1994 A major amily of motifs invoving GA mismatches in ribosomal RNA It Mol Biol 242 18 Gautheret D Konings D 8 Gutell R R 1995 GU basepairing motifs in ribosomal RNA RNA 1 807 814 Gehring K Leroy I L 8 Gueron M 1993 A tetra meric DNA structure with protonated cytosi necytosine basepairs Nature 363 561565 Gesteland R F 8 Atkins I F 1993 Editors of The RNA World Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Gesteland R F Cech T R 8 Atkins I F 1999 Editors of The RNA World 2nd edit Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Golden B L Gooding A R Podell E R 8 Cech T R 1998 A preorganized active site in the crystal structure of the Tetrahymem ribozyme Science 282 Gonzalez R L Ir 8 Tinoco L Ir 1999 Solution struc ture and thermodynamics of a divalent metal ion binding site in an RNA pseudoknot It Mol Biol 289 12671282 Greenbaum N L Radhakrishnan L Patel D I 8 Hirsh D 1996 Solution structure of the donor site of a transsplicing RNA Structure 4 725733 Grosjean H 8 Benne R 1998 Editors of Modi cation and Editing of RNA ASM Press Washington DC Hermann T 2000 Strategies for the design of drugs targeting RNA and RNAprotein complexes Angew Chem Int Edit in the press Hermann T 8 Westhof E 1998a Exploration of metal ion binding sites in RNA f lds by Brownian dynamics simulations Structure 6 1303 1314 Hermann T 8 Westhof E 1998b RNA as a drug tar get chemical modelling and evolutionary tools Curr Opin Biotechnol 9 66 73 Hermann T Auffinger P Scott W G 8 Westhof E 1997 Evidence for a hydroxide ion bridging two magnesium ions at the active site of the hammer head ribozyme Nucl Acids Res 25 34213427 Heus H A 8 Pardi A 1991 Structural features that give rise to the unusual stability of RNA hairpins containing GNRA loops Science 253 191194 Heus H A Wijm g S S Hoppe H 8 Hilbers C W 1997 The detailed structure of tandem GA mismatched base pair motifs in RNA duplexes is context dependent It Mol Biol 271 147158 Hilbers C W Michiels P I A 8 Heus H A 1998 New developments in structure determination of pseudoknots Biopolymers 48 137153 Holbrook S R 8 Kim S H 1997 RNA crystallogra phy Biopolymers Nucl Acids Sci 4 321 Holbrook S R Sussman I L Warrant R W Church G M 8 Kim S H 1977 RNAligand interactions L Magnesium binding sites in yeast tRNAPhe Nucl Acids Res 4 28112820 Holbrook S R Cheong C Tinoco L Ir 8 Kim S H 1991 Crystal structure of an RNA double helix incorporating a track of nonWatsonCrick base pairs Nature 353 579581 Holland I A 8 Hoffmann D W 1996 Structural fea tures and stability of an RNA triple helix in sol ution Nucl Acids Res 24 28412848 Hoogstraten C G Legault P 8 Pardi A 1998 NMR solution structure of the leaddependent ribozyme evidence for dynamics in RNA catalysis It Mol Biol 284 337350 Huang S Wang YX 8 Draper D E 1996 Structure of a hexanucleotide RNA hair in loop conserved in ribosomal RNAs It Mol Biol 258 308 321 Hud N V Smith F W Anet F 8 Feigon I 1996 The selectivity for K versus Naquot in DNA quadru plexes is dominated by relative free energies of hydration a thermodynamic analysis by 1H NMR Biochemistry 35 15383 15390 Ippolito I A 8 Steitz T A 1998 A 13 A resolution crystal structure HIV1 transactivation response region RNA stem reveals a metal ion dependent bulge conformation Proc Natl Acad Sci USA 95 98199824 Iack A Ladner I E Rhodes D Brown R S 8 Klug A 1977 A crystallographic study of metalbinding to yeast phenylalanine transfer RNA It Mol Biol 111 315328 Iaeger L Michel F 8 Westhof E 1994 Involvement of a GNRA tetraloop in longrange RNA tertiary interactions It Mol Biol 236 12711276 Iames B D Olsen G L Liu I 8 Pace N R 1988 The secondary structure of ribonuclease P RNA the catalytic element of a ribonucleoprotein enzyme Cell 52 1926 Iang S B Hung L W i Y L Holbrook E L Carter R I 8 Holbrook S R 1998 Structure of an RNA internal loop consiting of tan em A basepairs Biochemistry 37 11726 11731 Iiang L 8 Patel D I 1998 Solution structure of the tobramycinRNA aptamer complex Nature Struct Biol 5 769774 Iiang L Suri A K Fiala R 8 Patel D I 1997 Sac ideRNA recognition in an aminoglycoside anti bioticRNA aptamer complex Chem Biol 4 3550 Iiang L Majumdar A Hu W Iaishree T L Xu W 8 Patel D I 1999 Saccharide RNA recognition in a complex formed between neomycin B and an A a tamer Structure 7 817827 Iossinet F Paillart C Westhof E Hermann Skripkin T E Lodmell I S Ehresmann C Stitching Together RNA Tertiary Architectures Ehresmann B 8 Marquet R 1999 Dimerization of HIV1 genomic RNA of subtypes A and B RNA loop structure and magnesium binding RNA 5 12221234 Iucker F M 8 Pardi A 1995 GNRA loops make a Uturn RNA 1 219222 Iucker F M H Yi R R Moors E H 8 hydrogen bonds in GNRA tetraloops It Mol Biol 264 968 980 Kambach C Walke S 8 Nagai K 1999 Structure and assembly of the spliceosomal small nuclear ribonucleoprotein particles Curr Opin Struct Biol 9 222230 Kang C Zhang X Ratliff R Moyzis R 8 Rich A 1992 Crystal structure of fourstranded Oxytricha telomeric DNA Nature 356 126131 Kettani A Bouaziz S Gorin A Zhao H Iones R A 8 Patel D I 1998 Solution structure of a Na cat ion stabilized DNA quadruplex GGGG and GCGC tetrads formed b GGG C repeats observed in adeno associated viral DNA It Mol Biol 282 619636 Kieft I S 8 Tinoco I Ir 1997 Solution structure of a metal binding site in the ma or complexed with cobaltIII hexammine Structure 5 713 721 Kim SH Suddath F L Quigley G I McPherson A Sussman I L Wang A H Seeman N C 8 Rich A 1974 Three dimensional tertiary structure of yeast phenylalanine transfer RNA Science 185 435440 Klinck R Liquier Taillandier E Gouyette C Huynh inh T 8 Guittet E 1995 Structural characterization of an intramolecular RNA triple helix by NMR spectroscopy Eur It Biochem 233 544 553 Kolk M Heus H A 8 Hilbers C W 1997 The structure of the isolated central hairpin of the HDV antigenomic ribozyme novel structural features and similarity of the loop in the ribozyme and free in solution EMBO It 16 36853692 Kolk M van der Graaf M Wijmenga S S Pleij C W A Heus H A 8 Hilbers C W 1998 NMR structure of a classical pseudoknot interplay of single and doublestranded RNA Science 280 8 Kuimelis R G 8 McLaughlin L W 1998 Mechan isms of ribozyme mediated RNA cleavage Chem Rev 98 10271044 Laing L G Gluick T C 8 Draper D E 1994 Stabil ization of RNA structure by Mg ions Specific and nonspecific effects It Mol Biol 237 577587 Laughlan G Murchie A I Norman D G M Moody R C Lilley D M 8 Luisi B 1994 The highresolution crystal structure of a parallelstranded guanine tetraplex Science 265 20524 Lee A I 8 Crothers D M 1998 The solution struc re of an RNA looploop com lex the ColEl inverted loop sequence Structure 6 993 1005 Legault R Hoogstraten C G Metlitzky E 8 Pardi A 1998a Order dynamics and metalbinding in the leaddependent ribozyme It Mol Biol 284 325335 Legault R Li I Mogridge Kay L E 8 Greenblatt I 1998b NMR structure of the bacteriophage 9 N peptideboxB RNA complex recognition of a GNRA fold by an arginine rich motif Cell 93 289 299 Moore Lehnert V Iaeger L Michel F 8 Westhof E 1996 New looploop tertiary interactions in selfsplicing introns of subgroup IC and ID a complete 3D model of the Tetrahymem thermophila ribozyme Chem Biol 3 993 1009 Leonard G A McAuleyHecht K E Ebel S Lou h M Brown T 8 Hunter W N 1994 Crystal and molecular structure of rCGCGAAUUAGCG duplex containing two GantiAanti basepairs Structure 2 483494 Leontis N B 8 Westhof E 1998a The 5 S rRNA loop E chemical probing and phylogenetic data versus crystal structure RNA 4 1134 1153 Leontis N B 8 Westhof E 1998b A common motif organizes the structure of multihelix loops in 16 S and 23 S ribosomal RNAs It Mol Biol 283 571583 Lietzke S E Barnes C L Berglund I A 8 Kundrot C E 1996 The structure of an RNA dodecamer show how tandem UU base pairs increase the range of stable RNA structures and the diversity of recognition sites Structure 4 917930 Lilley D M I 1999 Structure folding and catalysis of e small nucleolytic ribozymes Curr Opin Struct Biol 9 330338 Malhotra A 8 Harvey S A 1994 A quantitative model of the Escherichai coli 16 S rRNA in the 30 S ribosomal subunit It Mol Biol 240 308340 Marino I R Gregorian R S Ir Csankovszki G 8 Crothers D M 1995 Bent helix formation between RNA hairpins with complementary loops Science 268 1448 1454 Masquida B 8 Westhof E 1999 Crystallographic structures of RNA oligoribonucleotides an ri 0 es In Oxford Handbook of Nucleic Acid Structure Neidle S ed pp 533 565 Oxford University Press Oxford Masquida B Felden B 8 Westhof E 1997 Context dependent RNARNA recognition in a threedimen sional model of the 16 S rRNA core Bioorg Med Chem 5 10211035 Massire C Iaeger L 8 Westhof E 1998 Derivation of the threedimensional architecture of bacterial ribonuclease P RNAs from comparative sequence analysis It Mol Biol 279 773 793 McKay D B 8 Wedekind I E 1999 Small ribozymes The role of metal ions in RNA biochemistry In The RNA World Gesteland R R Cech T R 8 Atkins I R eds 2nd edit pp 265286 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Michel F 8 Westhof E 1990 Modelling of the three dimensional architecture of group I introns based on comparative sequence analysis It Mol Biol 216 585610 Michel F 8 Westhof E 1996 Visualizing the logic ehind RNA selfassembly Science 273 1676 1677 Moore R B 1998 The three dimensional structure the ribosome and its components Annu Rev Biophys Biomoll Structl 27 3558 Moore R B 1999 Structural motifs in RNA Annu Rev Biochem 67 287300 Mueller F 8 Brimacombe R 1997 A new model for threedimensional folding of Escherichia coli 16 S ribosomal RNA It Mol Biol 271 524 544 Mueller U Schubel H Sprinzl M 8 Heinemann U Crystal structure of acceptor stem of tRNAAla from Escherichia coli shows unique GU wobble basepair at 1 16 A resolution RNA 5 670 677 848 Stitching Together RNA Tertiary Architectures Murray I B Terwey D P Maloney L Karpeisky A Usman N Beigelman L 8 Scott W G 1998 The structural basis of hammerhead ribozyme self cleavage Cell 92 665673 Nowakowski I 8 Tinoco L Ir 1999 RNA structure in solution In Oxford Handbook of Nucleic Acid Struc ture Neidle S ed pp 567602 Oxford University Press Oxford Pan T 8 Uhlenbeck O C 1992 A small metalloribo Zyme with a twostep mechanism Nature 358 560 5 3 6 Pan 3 Mitra S N 8 Sundaralingam M 1998 Structure of a 16 mer RNA duplex rGCAGA CUUAAAUCUGC2 with wobble CA mis matches It Mol Biol 283 977984 Pan 3 Mitra S N 8 Sundaralingam M 1999 Crystal structure of an RNA 16 mer duplex rGCAGAGUUAAAUCUGC2 with nonadjacent GsynAanti mispairs Biochemistry 38 2826 2831 Patel D I 1997 Structural analysis of nucleic acid aptamers Curr Opin Chem Biol 1 3246 Patel D I 1999 Adaptive recognition in RNA com plexes with peptides and protein modules Curr Opin Struct Biol 9 74 87 Patel D L Suri A K Iiang F Iiang L Fan P Kumar R A 8 Nonin S 1997 Structure recog nition and adaptive binding in RNA aptamer com plexes It Mol Biol 272 645664 Pleij C W Rietveld K 8 Bosch L 1985 A new prin ciple of RNA folding based on pseudoknotting Nucl Acids Res 13 17171731 Pley H W Flaherty K M 8 McKay D B 1994a Threedimensional structure of a hammerhead ribo Zyme Nature 372 68 74 Pley H W Flaherty K M 8 McKay D B 1994b Mo el for an RNA tertiary interaction from the structure of an intermolecular complex between a GAAA tetraloop and an RNA helix Nature 372 111113 Portmann S Grimm S orkman C Usman N 8 Egli M 1996 Crystal structure of an Aform duplex with singleadenosine bulges and a confor mational basis for sitespecific RNA selfcleavage Chem Biol 3 173 184 Puglisi I D 8 Williamson I R 1999 RNA interaction with small ligands and peptides In The RNA World Gesteland R F Cech T R 8 Atkins I F eds 2nd edit pp 403425 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Puglisi I D Wyatt I R 8 Tinoco L Ir 1988 A pseu doknotted RNA oligonucleotide Nature 331 283 286 Puglisi I D Chen L Blanchard S 8 Frankel A D 1995 Solution structure of a bovine immunodefi ciency virus TatTAR peptideRNA complex Science 270 12001203 Pyle A M 1993 Ribozymes a distinct class of metal loenzymes Science 261 709714 Quigley G I 8 Rich A 1976 Structural domains of transfer RNA molecules The ribose 2 hydroxyl which distinguishes RNA from DNA plays a key role in stabilizing tRNA structure Science 194 796 6 80 Quigley G L Teeter M M 8 Rich A 1978 Struc tural analysis of spermine and magnesium ion bind ing to yeast phenylalanine transfer RNA Proc Natl Acad Sci LISA 75 6468 Rajagopal P 8 Feigon I 1989 Triple strand formation 39 e o urinehomopyrimidine DNA oligonu cleotides dGA4 and dTC4 Nature 339 637640 Rife I P 8 Moore P B 1998 The structure of a meth ylated tetraloop in 16 S ribosomal RNA Structure 6 747756 Robertus I D Ladner I E Finch I T Rhodes D Brown R S Clark B F C 8 Klug A 1974 Structure of yeast phenylalanine tRNA at 3 A resol ution Nature 250 546551 Saenger W 1984 Principles of Nucleic Acid Structure pringer New York Schindelin H Zhang M Bald R Furste I P Erdmann V A 8 Heinemann U 1995 Crystal structure of an RNA dodecamer containing the Escherichia coli Shine Dalgarno sequence It Mol Biol 249 595603 Schmitz U Iames T L Lukavsky P 8 Walter P 1999 Structure of the most conserved internal loop in SRP RNA Nature Struct Biol 6 634 638 Scott E C 8 Uhlenbeck O C 1999 A re investigation of the thio effect at the hammerhead cleavage site Nucl Acids Res 27 479484 Scott W G Finch I T 8 Klug A 1995 The crystal structure of an allRNA hammerhead ribozyme a pro osed mechanism for RNA catalytic cleavage Cell 81 9911002 Scott W G Murray I 13 Arnold I R P Stoddard B L 8 Klug A 1996 Capturing the structure of a catalytic RNA intermediate the hammerhead ribo e Science 274 20652069 Smith D 8 Pace N R 1993 Multiple magnesium ions in the ribonuclease P reaction mechanism Bio chemistry 32 5273 5281 Sontheimer E L Gordon P M 8 Piccirilli I A 1999 Metal ion catalysis during group II intron selfspli cing parallels with the spliceosome Genes Dev 13 17291741 Steitz T A 1999 RNA recognition by proteins In The RNA World Gesteland R F Cech T R 8 Atkins I F eds 2nd edit pp 427450 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Steitz T A 8 Steitz I A 1993 A general twoion mechanism for catalytic RNA Proc Natl Acad Sci USA 90 6498 6502 Streicher B Westhof E 8 Schroeder R 1996 The environment of two metal ions surrounding the splice site of a group I intron EMBO It 15 2556 2564 Strobel S A 1999 A chemogenetic approach to RNA function structure analysis Curr Opin Struct Biol 9 346 352 Strobel S A 8 Doudna I A 1997 RNA seeing double close packing of helices in RNA tertiary structure Trends Biochem Sci 22 262266 of a ribosomal frameshifting viral pseudoknot Nature Struct Biol 6 285292 Szewczak A A 8 Moore P B 1995 The sarcinricin loop a modular RNA It Mol Biol 247 8198 Szewczak A A Moore P 3 Chang Y L 8 Wool L G 1993 The conformation of the sarcinricin loop from 28 S ribosomal RNA Proc Natl Acad Sci USA 90 95819585 Tuerk C Gauss P Thermes C Groebe D R Gayle Guild M Stormo G D AubentonCarafa Y Uhlenbeck O C 8 Tinoco L Ir 1988 CUUCGG hairpins extraordinarily stable RNA secondary Stitching Together RNA Tertiary Architectures 849 structures associated with various biochemical pro cesses Proc Natl Acad Sci LISA 85 13641368 Valegard K Murray I B Stockley P G Stonehouse N I 8 Liljas L 1994 Crystal structure of an RNA bacteriophage coat proteinoperator complex Nature 371 623626 Varani L Hasegawa M Spillantini M G Smith M L Murrell I R Ghetti B Klug A Goedert M 8 Varani G 1999 Structure of tau exon 10 splicing regulatory element RNA and destabilization by mutations of frontotemporal dementia and rkin sonism linked to chromosome 17 Proc Natl Acad sci USA 96 82298234 VianiPuglisi E Green R Noller H F 8 Puglisi I D 1997 Structure of a conserved RNA component of the peptidyl transferase center Nature Struct Biol 4 7 78 Wang Y X Lu M 8 Draper D E 1993 Specific ammonium ion requirement for functional riboso mal RNA tertiary structure Biochemistry 32 12279 282 Wedekind I E 8 McKay D B 1999 Crystal structure of a leaddependent ribozyme revealing metal bind ing sites relevant to catalysis Nature Struct Biol 6 261268 Weeks K M 8 Crothers D M 1993 Major groove accessibility of RNA Science 261 15741577 Westhof E 1988 Water an integral part of nucleic acid structure Annu Rev Biophys Biophys Chem 17 125144 Westhof E 8 Michel F 1998 Ribozyme architectural diversity made visible Science 282 251252 Westhof E Dumas P 8 Moras D 1985 Crystallo graphic refinement of yeast aspartic acid transfer RNA1 Mol Biol 184 119145 Received 18 August 1999 received in Westhof E Dumas P 8 Moras D 1988 Hydration of transfer RNA molecules a crystallographic study Biochimie 70 145165 Wimberly B Varani G 8 Tinoco L Ir 1993 The con formation of loop E of eucaryotic 5 S ribosomal RNA Biochemistry 32 10781087 Wimberly B T Guymon R McCutcheon I P White S W 8 Ramakrishnan V 1999 A detailed view of a ribosomal active site the structure of the L11 RNA complex Cell 97 491502 Woese C R Winker S 8 Gutell R R 1990 Architec ture of ribosomal RNA constraints on the sequence of tetraloops Proc Natl Acad Sci LISA 87 8467 8471 Wyatt I R 8 Tinoco L Ir 1993 RNA structural elements and RNA function In The RNA World Gesteland R F 8 Atkins I F eds pp 465496 Cold Spring Harbor Laboratory Press Cold Spring Harbor NY Wyatt I R Puglisi I D 8 Tinoco L Ir 1990 RNA pseudoknots stability and loop size requirements It Mol Biol 214 455470 Ye X Kumar R A 8 Patel D I 1995 Molecular rec ition in the bovine immunodeficiency virus Tat peptideTAR RNA complex Chem Biol 2 827840 Ye X Gorin A Ellin on A D 8 Patel D I 1996 Deep penetration of an ochelix into a widened RNA major groove in the HIV1 rev eptideRNA apta mer complex Nature Struct Biol 3 10261033 Yonath A 8 Franceschi F 1998 Functional universal ity and evolutionary diversity insights from the structure of the ribosome Structure 6 679684 Zimmermann R Ienison D W1C L Simorre P 8 Pardi A 1997 Interlocking struc tural motifs mediate molecular discrimination y a theophyllinebinding RNA Nature Struct Biol 6 49 Edited by P E Wright revised form 14 October 1999 accepted 14 October 1999