comparative analysis, is based on a very simple andprofound principle. This method has been utilized to predictthe secondary structure and the early stages of the tertiarystructure of several RNA molecules, including the rRNAs.In addition to these structure predictions, the comparativeapproach has also revealed new information about RNAstructural motifs and other principles of RNA structure.Inferring higher-order structure from patternsof sequence variationShortly after the first tRNA sequence was determined ,it was rationalized from a comparative perspective that alltRNA sequences should have equivalent secondary andtertiary structures to allow them to interact with the samebinding sites on the ribosome and with the same set ofproteins and RNAs during protein synthesis. Two basicprinciples form the foundation for the comparative analysisof RNA structure: firstly, different RNA sequences canfold into the same secondary and tertiary structures and,secondly, the unique structure and function of an RNAmolecule is maintained through the evolutionary processof mutation and selection. We utilized this comparativeparadigm for the prediction of the 16S and 23S rRNAstructures. We assumed that all 16S (and 16S-like) and 23S(and 23S-like) rRNAs have the same general secondary andtertiary structures, regardless of the extent of conservationand variation among the sequences. The correct helicesthat have been identified using comparative analysis arepresent in the same homologous region of the rRNAs andhave variation in the composition of the sequences, whilstmaintaining G•C, A•U and G•U base pairs. Initially, weidentified base-paired positions within a potential helix thathave ‘covariation’ (similar patterns of variation) in a set ofsequences aligned for maximum sequence identity [8–10].Proposed helices with two or more covariations wereconsidered ‘proven’. Versions of the 16S and 23S rRNAstructure models from the early 1980s (Santa Cruz/Urbanaversions) are shown in Figure 1. The majority of the helicesin these early structure models had at least one covariationper helix. We considered this model to be the minimalstructure, that is, there were areas that were incomplete.Two other sets of 16S and 23S rRNA structure modelswere determined independently with comparative methods[11–14], whereas another set of model diagrams was adaptedin full from previously proposed structure models [15–17].Subsequently, as the number of sequences in our 16S and23S rRNA alignments surpassed 25, we developed differentalgorithms and computer programs to identify positions inan alignment that have similar patterns of variation [18–20].Given this series of improvements in the covariationalgorithms, coupled with very dramatic increases in the302 Nucleic acidsFigure 1IIIIII501001502002503003504004505005506006507007508008509009501000105011001150120012501300135014001450150015501600164029005’ 3’3’ half1050100150200250300350400450500550600650700750800850900950100010501100115012001250130013501400145015005’3’IIIIIIIVVVI5’3’165017001750180018501900195020002050210021502200225023002350240024502500255026002650270027502800285029005’ half(a) (b) (c)Current Opinion in Structural BiologyThe original (1980–81) Noller-Woese-Gutell comparative structuremodels for the 16S and 23S rRNAs. (a) 16S rRNA (adapted from). (b) 23S rRNA, 5′ half (adapted from ). (c) 23S rRNA, 3′ half(adapted from ). E. coli (GenBank accession number J01695) isused as the reference sequence. Each of these models has beensuperimposed onto the corresponding current model diagrams tohighlight the similarities and differences. Nucleotides are replaced withcolored dots: black, positions that are unchanged between the originaland current models; blue, base pairs present in the original modelsbut absent from the current models; red, positions that are unpaired inthe original models but are part of a base pair in the current models;green, positions that are part of one base pair in the original modelsbut are part of a different base pair in the current models. Full-pageversions of each panel are available online athttp://www.rna.icmb.utexas.edu/ANALYSIS/COSB2002/ (part of theCRW site at http://www.rna.icmb.utexas.edu/).
number and diversity of rRNA sequences in our sequencecollection, we were able to identify more positions withsimilar patterns of variation. Although the early covariationanalysis only identified those covariations that involve A•Uand G•C pairings within a potential helix, our algorithmshave, for the past ten years, identified all positionalcovariations, regardless of base pair type and their types ofinterchanges with other base pairs (e.g. U•U ↔ C•C,A•A ↔ G•G, U•U ↔ G•G), and independent of the spatialrelationship with other base pairings and structural elements. Consequently, we began identifying single base pairingsnot flanked by other base pairings, noncanonical base pairsand other types of tertiary interactions (see below). Inaddition to the inclusion of newly identified base pairs,previously proposed base pairs were removed from thestructure models when the ratio of covariation to variationdropped with increasing numbers of sequences.To gauge the extent of positional covariation and ourconfidence in the accuracy of each of these proposed basepairs, we established a quantitative scoring method.Higher scores reflect a greater extent of pure covariation(simultaneous changes at both of the paired positions),larger numbers of exchanges between a set of base pairtypes that covary with one another (e.g. A•U ↔ G•C)and/or a larger number of mutual changes or covariationsthat occur during the evolution of the RNA (also calledphylogenetic events). These three parameters can,individually or collectively, influence our confidence in aputative base pair. For example, we were more confidentin the authenticity of the 570•866 base pair in 16S rRNAbecause of several phylogenetic events within the bacteria,archaea and eucarya . These 16S and 23S rRNAcovariation-based structure models only contain those basepairs with positional covariation or G•C, A•U or G•U basepairs that are within a regular helix and present in morethan 80% of the sequences.The most recent comparative structure models for 16S and23S rRNA are shown in Figure 2 and are based on theanalysis of approximately 7000 16S and 1050 23S rRNAsequences [21,23]. These two structure models are theculmination of 20 years of comparative analysis (seebelow). The base pair symbols are color coded to reveal ourconfidence in the authenticity of that base pair; base pairswith the highest covariation scores are shown in red,followed by green and black. Base pairs with gray symbolsare conserved in more than 98% of the sequences, whereasRibosomal RNA comparative structure models Gutell, Lee and Cannone 303Figure 2IIIIII501001502002503003504004505005506006507007508008509009501000105011001150120012501300135014001450150015501600164029005’ 3’3’ half(2407-2410)(2010-2011)(2018)(2057/2611 BP)(2016-2017)AIVVVI5’3’165017001750180018501900195020002050210021502200225023002350240024502500255026002650270027502800285029005’ half(1269-1270)(413-416)(1262-1263)(746)(531)1050100150200250300350400450500550600650700750800850900950100010501100115012001250130013501400145015005’3’IIIIIIA(a) (b) (c)Current Opinion in Structural BiologyThe current Noller-Woese-Gutell comparative structure models for the16S and 23S rRNAs. (a) 16S rRNA. (b) 23S rRNA, 5′ half. (c) 23SrRNA, 3′ half. E. coli (GenBank accession number J01695) is used asthe reference sequence. Nucleotides are replaced with colored dotsthat represent confidence in the base pair: red, high covariation scores;green, lower but significant covariation scores occurring within astandard helix containing a red base pair; black, even lower covariationscores occurring within a standard helix containing a red base pair;gray, conserved in more than 98% of the sequences occurring withina standard helix containing a red base pair; blue, do not have a significantamount of pure covariation and do not occur within a standard helix (see for additional details). Base pair symbols indicate the type of basepair: line, canonical base pair; small closed circle, G•U base pair; largeopen circle, G•A base pair; large closed circle, other noncanonicalbase pairs. Nucleotides involved in tertiary interactions (includingpseudoknots) are boxed and connected with lines. Diagrams adaptedfrom . Full-page versions of each panel are available online at theCRW site (http://www.rna.icmb.utexas.edu/ANALYSIS/COSB2002/).
blue base pairs do not have a significant amount of purecovariation and do not occur within a standard helix(see  for more details). As the majority of the base pairshave red symbols, we believe that nearly all of the basepairs in the current 16S and 23S rRNA covariation-basedstructure models are correct (see below).The evolution of the 16S and 23S rRNA covariation-basedstructure models is shown graphically in Figure 1 andquantitatively in Table 1. To allow easy comparison with thecurrent models, the original 1980–81 16S and 23S rRNAstructure models were redrawn using the current models asa template (Figure 1). Base pairs that are present in both theoriginal and current models are shown in black, and thosethat are different in the original structure models and themost recent covariation-based structure models are illustratedin blue, red and green. Blue base pair symbols indicate basepairs in the original models that are absent from the currentmodels, red nucleotides are unpaired in the original modelsand paired in the current models, and green nucleotides arepart of different base pairs in the two structure models.In 1980–81, the 16S and 23S rRNA structure models werebased on just two complete rRNA sequences per structure;at the end of 1999, this work culminated with the analysis ofapproximately 7000 16S and 1050 23S rRNA sequences.These structure models evolved over nearly 20 years as thecollection of sequences grew and our methods to identifyand score covariations were developed and refined. To assessthe changes, the original 1980–81 structure models werecompared with the current 1999 structure models (Table 1,adapted from Section 1b on the ‘Comparative RNA Web’[CRW] site and database; http://www.rna.icmb.utexas.edu).We draw four significant conclusions from this analysis.Firstly, nearly 60% of the base pairs in the current 16SrRNA structure model were predicted from the analysisof two sequences for the original structure model; nearly78% of the current 23S rRNA base pairings were predictedfrom the original structure model. Secondly, in contrast,approximately 80% of the original 16S and 87% of theoriginal 23S rRNA base pairs proposed in 1980–81 arepresent in the current models. Thirdly, approximately 7016S and 100 23S initial base pairs have been removed fromthe original rRNA structure models. Finally, the number ofunusual, tertiary and tertiary-like base pairings that are pre-dicted with confidence increases in parallel with increasesin the number and diversity of rRNA sequences studiedand with improvements in the covariation algorithms. Inconclusion, the major components of the 16S and 23SrRNA structure models were predicted correctly from theanalysis of just a few 16S and 23S rRNA sequences that areapproximately 75% similar to one another. Thousands ofadditional rRNA sequences with significant degrees ofsimilarity and diversity with one another were subsequentlyanalyzed with covariation analysis to refine the secondarystructure models, to begin to identify tertiary base pairs andto establish a system to measure the extent of covariation atall of the proposed base pairs. Beyond the prediction ofbase pairs with covariation analysis, the comparativesequence and structure data are encrypted with fundamentalprinciples of RNA structure and archaeological markersthat indicate the ancestry of that RNA sequence .Our next task is to decipher these ‘treasures’ from thecomparative RNA sequence and structure data sets. Tothis end, we have established the CRW site and database(; http://www.rna.icmb.utexas.edu/) to organize, analyzeand disseminate comparative data for the 5S, 16S (and16S-like) and 23S (and 23S-like) rRNAs, group I and IIintrons, and tRNAs. The main types of information anddata available online for each of these RNAs are: the currentcomparative RNA structure model; nucleotide and basepair frequency tables for all positions in the referencestructures; secondary structure conservation diagrams thatreveal the extent of conservation of the RNA sequenceand structure; more than 400 representative secondarystructure diagrams for organisms from groups that span thephylogenetic tree and reveal the major forms of structuralvariation; nearly 12,000 publicly available sequences thatare 90% or more complete; and sequence alignments.304 Nucleic acidsTable 1Summary of the evolution of the Noller-Woese-Gutell 16S and 23S rRNA structure models from the first to the most recentcovariation-based structure models (adapted from Table 3a,b in ).Model 16S rRNA 23S rRNADate 1980 1999 1981 19991. Approximate number of complete sequences 2 7000 2 10502. Percentage of 1999 sequences* 0.03 100 0.2 1003. Number of bp proposed correctly* 284 478 676 8704. Number of bp proposed incorrectly* 69 0 102 05. Total bp in model (3 + 4) 353 478 778 8706. Percentage of bp in model present in the current model (3 / X)*†59.4 100 77.7 1007. Accuracy of proposed bp (3 / 5) 80.5 100 86.9 1008. Number of bp in current model missing from this model (X – 3)*†194 0 194 09. Number of tertiary bp proposed correctly* 4 40 4 6510. Percentage of tertiary bp proposed correctly* 10.0 100 6.2 10011. Number of base triples proposed correctly* 0 6 0 712. Percentage of base triples proposed correctly* 0 100 0 100*Comparisons are made against the current (1999) models. †X = 478 for 16S rRNA; X= 870 for 23S rRNA. bp, base pairs.
This type of comparative data is the foundation for thesubsequent identification and analysis of RNA structuralmotifs. Although the patterns of variation at both positionsin many of the base pairs in the RNA structure are similarand thus should be identified with covariation analysis,other sets of base pairs do not have similar patterns ofvariation at the two interacting positions. Thus, one of thelarger goals of comparative analysis is to predict those basepairs lacking similar patterns of variation that occur inseveral different types of structural elements, as well asthose base pairs with positional covariation that are conservedamong the sequences in that data set. The process ofcomparative analysis, then, is to first predict base pairingswith covariation analysis, followed by the identification ofmotifs that are composed of unique arrangements ofsequences within specific structural elements. SeveralRNA structural motifs have been identified and/or are stillbeing defined from sequence and structure perspectives.These motifs include:1. Unpaired adenosines in the covariation-based structuremodel [18,25•].2. Tetraloops — hairpin loops with four nucleotides that arecomposed of specific sequences .3. Tetraloop receptors and other tertiary interactions involvingtetraloops [27–30].4. Dominant G•U base pairs [31,32].5. Tandem G•A oppositions [33,34].6. Base triples .7. Adenosine platforms [25•,35].8. U-turns .9. E loops (or S turns) [25•,37,38].10. E-like loops [25•].11. Cross-strand purine stacks .12. A•A and A•G oppositions/base pairs at the ends ofhelices [10,40,41•].13. Lone pair triloops (; RR Gutell et al., unpublisheddata).14. A-minor motif [42•,43•].15. Kink-turn [44•].Crystal structures of the 16S and 23S rRNAs:the accuracy of the rRNA comparativestructure modelsTo assess the accuracy of the covariation-based structuremodels, the comparative models for tRNA [19,20,45–50],fragments of 5S rRNA , the L11-binding region of23S rRNA [9,21,23] and the group I intron [52,53] werecompared with the corresponding high-resolution crystalstructures [39,54–58]. Nearly all of the secondary structurebase pairings and a few of the tertiary base pairs observedin the crystal structure were predicted in the comparativestructure models for all of these RNAs. More recently, thehigh-resolution crystal structures of the 30S [59••,60] and50S [61••] ribosomal subunits were solved, giving us theopportunity to evaluate the accuracy of our most recent16S and 23S rRNA structure models. The results wereagain affirmative: approximately 97–98% of the basepairings predicted with covariation analysis (in the finalcovariation-based structure models) are indeed presentin the 16S and 23S rRNA crystal structures (Table 2;RR Gutell et al., unpublished data). The accuracy of the 16Sand 23S rRNA covariation-based structure prediction notonly augments the credibility of the comparative approach,but it also validates the sequence alignments that havebeen initiated, refined and expanded over the past 20 years,the initial covariation analysis and our subsequentRibosomal RNA comparative structure models Gutell, Lee and Cannone 305Table 2Comparison of the current comparative structure models and the crystal structures of the 16S and 23S rRNAs*.16S rRNA†23S rRNA‡TotalPredicted base pairs§Model CB #461 / 476 / 97% 779 / 797 / 98% 1240 / 1273 / 97%Tentative CB#8 / 23 / 35% 18 / 36 / 50% 26 / 59 / 44%Motif-based¶45 / 65 / 70% 86 / 122 / 70% 131 / 187 / 70%Crystal structure interactions¥+/+ base–base 514 883 1397–/+ base–base 56 425 481Total base–base 683 1297 1862Base–backbone 49 237 286*A more complete analysis will be presented later (RR Gutell et al., unpublished data). †T. thermophilus, GenBank accession number M26923,PDB code 1FJF . ‡H. marismortui, GenBank accession number AF034620, PDB code 1JJ2 . §Data are shown as approximatenumber of base pairs present in the crystal structure / approximate number of predicted base pairs / percentage of predicted base pairspresent in the crystal structure. #CB, covariation-based. ¶The motifs analyzed here are AA.AG@helix.ends , tandem GA [33,34], E andE-like loops , lone pair triloops (RR Gutell et al., unpublished data) and base triples . ¥Approximate numbers of interactions in the tworibosomal crystal structures.
covariation algorithms and their refinements. In additionto the final covariation-based structure model, nearly 45%of the tentative covariation-based base pairs and 70% ofthe motif-based base pairs that were predicted are in thecrystal structure (Table 2). In total, about 90% of the basepairs predicted by comparative analysis are from thecovariation-based analysis and 10% are from the alternativemotif-based analysis ([20,25•,33,34,41•]; RR Gutell et al.,unpublished data).The secondary structure diagrams for Thermus thermophilus16S rRNA and Haloarcula marismortui 23S rRNA are shownin Figure 3. All of the base–base and base–backboneinteractions in the 30S [59••] and 50S [61••] ribosomalsubunit crystal structures are colored to reflect the initialidentification of each pairing. The three primary categoriesare: present in both the comparative model (covariationand motif analysis) and the crystal structure (+/+), presentin the comparative model but not in the crystal structure(+/–), and not present in the comparative model butpresent in the crystal structure (–/+). The nucleotides andbase pair symbols are colored red for +/+, green for +/–,blue for –/+ base–base interactions and brown for –/+base–backbone interactions.The affirmative base pairs that were predicted usingcovariation analysis (see red nucleotides and base pairsymbols in Figure 3) include: essentially all base pairs that arestrictly homologous between the E. coli reference structuremodels and the T. thermophilus 16S and H. marismortui 23SrRNA crystal structures that have a significant amount ofpositional covariation; base pairs that are standardWatson–Crick (G•C and A•U) and G•U base pairexchanges; base pairs that occur within standard secondarystructure helices (2 base pairs in length) that are nested(i.e. not a pseudoknot); individual base pairs and helices306 Nucleic acidsFigure 3Comparison of the current Noller-Woese-Gutellcomparative structure models for the 16S and23S rRNAs with the corresponding ribosomalsubunit crystal structures. (a) 16S rRNAversus the T. thermophilus structure(GenBank accession number M26923;PDB code 1FJF; [59••]). (b) 23S rRNA,5′ half versus the H. marismortui structure(GenBank accession number AF034620;PDB code 1JJ2; [61••]). (c) 23S rRNA, 3′ halfversus the H. marismortui structure (GenBankaccession number AF034620; PDB code 1JJ2;[61••]). Nucleotides are replaced with coloreddots that show the sources of theinteractions: red, present in both thecovariation-based structure model and thecrystal structure; green, present in thecomparative structure and not present inthe crystal structure; blue, not present inthe comparative structure and present in thecrystal structure; magenta, present in thecovariation-based tentatives or motif-basedanalysis, and present in the crystal structure;brown, base–backbone orbackbone–backbone interactions; purple,positions that are unresolved in the crystalstructure. Colored open circles aroundpositions show the third nucleotide of basetriples and colored open rectangles show thebase pairs of base triples. Colored open squaresare used for clarity. Full-page versions of eachpanel are available online at the CRW site(http://www.rna.icmb.utexas.edu/ANALYSIS/COSB2002/).5’3’50100150200250300350400450 50055060065070075080085090095010001050110011501200125013001350140014501500Current Opinion in Structural Biology(a)
that form pseudoknots, including tertiary interactions;lone pairs, including those in the lone pair triloop motif(RR Gutell et al., unpublished data); and noncanonicalbase pairs and their exchanges — A•A ↔ G•G, U•U ↔ C•C,A•G ↔ G•A, A•C ↔ G•U, U•A ↔ G•G, A•C ↔ U•A andA•G ↔ R•U .Although more than 1250 base pairs predicted with covari-ation analysis are in the crystal structure, approximately 35of them are not (see green nucleotides in Figure 3; notethat the green interactions include those predicted withboth covariation analysis and motif-based analysis). Themajority of these +/– proposed covariation-based base pairsthat are absolutely homologous between the E. coli referencemodels and the T. thermophilus 16S and H. marismortui 23SrRNA structures were not predicted with our highest (red)confidence rating. Instead, there was either no positionalcovariation or an insignificant amount of these putativebase pairs; these interactions were included in the structuremodel because they form a G•C, A•U or G•U pair in morethan 80% of the sequences and were adjacent to a base pairwith covariation. The majority of these +/– base pairs arecolored black, our lowest covariation confidence rating.The aberrant base pairs that are truly homologous betweenthe crystal structure and the E. coli reference structurehave two other important characteristics. First, all of theseputative base pairs occur at the ends of helices and, second,there is a bias in the types of base pairs that are not predictedcorrectly at the ends of helices. The two most frequentpairing types (in this latter category) are U•G and U•A(where the U is at the 5′ half of the helix). These putativebase pairs might not occur in the rRNA structure or,alternatively, they might be dynamic and are paired atcertain stages of protein synthesis and not in the states ofthe crystal structures analyzed here. There is a precedentfor conformational changes of the base pairings at the endsof helices. Positions 1408 and 1493 form an A•A base pairin the uncomplexed 30S ribosomal subunit (PDB code1FJF; [59••]), but are not paired when tRNA and mRNAare complexed to the 30S subunit . We speculate thatother A•A and A•G oppositions/base pairs at the ends ofhelices in the 16S and 23S rRNAs might be involved inconformational changes [41•]. There is also an interestinganecdote about the putative U•A pairings that are not inthe crystal structure. The orientation of these U•A pairswould place the conserved, ’unpaired’ adenosine at the3′ end of the loop, a very common arrangement in the 16Sand 23S rRNAs [25•].We will not know all of the structural possibilities for theseputative base pairings until we obtain more crystallographic,NMR or other experimental data for these regions of therRNA. Although comparative analysis has predictedapproximately 510 16S and 880 23S rRNA base pairs, anadditional ~170 16S and ~415 23S rRNA base pairs(base–base) are in the crystal structure that were notpredicted with comparative methods. Essentially, none ofthese ‘–/+’ base pairs has a significant amount of positionalcovariation and thus could not be predicted with covariationanalysis. In general, these ‘–/+’ base pairs comprisenoncanonical base pairs that are not associated withstandard helices that were predicted with covariationanalysis. A more detailed comparison between the compar-ative and crystal structures will be presented elsewhere(RR Gutell et al., unpublished data).ConclusionsCovariation analysis has accurately predicted all of thestandard secondary structure base pairings and helices inthe 16S and 23S rRNA crystal structures. These methodshave also identified some of the 16S and 23S rRNA tertiarybase–base interactions. Motif-based analysis has begun toidentify some of the base pairs that do not have similarpatterns of variation. Our future goal is to gain a betterunderstanding of tertiary base–base interactions from acomparative perspective and, more specifically, to determinetheir base pair types and exchanges, and the types ofstructural elements or motifs with which they are associated.A more complete set of RNA structure constraints isnecessary to accurately and reliably predict an RNA structurefrom its underlying sequence, and to understand thedynamics between structure and function.AcknowledgementsThis work was supported by the National Institutes of Health (GM48207),by the Welch Foundation (F-1427) and by start-up funds from the Institutefor Cellular and Molecular Biology at the University of Texas at Austin.References and recommended readingPapers of particular interest, published within the annual period of review,have been highlighted as:• of special interest••of outstanding interest1. Zuker M: On finding all suboptimal foldings of an RNA molecule.Science 1989, 244:48-52.2. 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