Gutell 071.jmb.2000.300.0791

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Gutell 071.jmb.2000.300.0791

  1. 1. Predicting U-turns in Ribosomal RNA withComparative Sequence AnalysisRobin R. Gutell1*, Jamie J. Cannone1, Danielle Konings2and Daniel Gautheret31Institute for Cellular andMolecular Biology, Universityof Texas at Austin, 2500Speedway, Austin, TX 78712-1095, USA2Department of MolecularCellular and DevelopmentalBiology, University of ColoradoCampus Box 347, BoulderCO 80309-0347, USA3Structural and GeneticInformation, CNRS UMR1889, 31 chemin JosephAiguier, 13 402, MarseilleCedex 20, FranceThe U-turn is a well-known RNA motif characterized by a sharp reversalof the RNA backbone following a single-stranded uridine base. In exper-imentally determined U-turn motifs, the nucleotides 3Hto the turn are fre-quently involved in tertiary interactions, rendering this motif particularlyattractive in RNA modeling and functional studies. The U-turn signatureis composed of an UNR sequence pattern ¯anked by a Y:Y, Y:A(Y ˆ pyrimidine) or G:A base juxtaposition. We have identi®ed 33 poten-tial UNR-type U-turns and 25 related GNRA-type U-turns in a large setof aligned 16 S and 23 S rRNA sequences. U-turn candidates occur inhairpin loops (34 times) as well as in internal and multi-stem loops (24times). These are classi®ed into ten families based on loop type, sequencepattern (UNR or GNRA) and the nature of the closing base juxtaposition.In 13 cases, the bases on the 3Hside of the turn, or on the immediate 5Hside, are involved in tertiary covariations, making these sites strong can-didates for tertiary interactions.# 2000 Academic PressKeywords: ribosomal RNA; comparative sequence analysis; U-turns;tertiary interactions*Corresponding authorIntroductionU-turns are small RNA structural motifs thatwere ®rst discovered in the anticodon and TcC-loop of tRNA (Quigley & Rich, 1976) and lateridenti®ed in the hammerhead ribozyme (Pley et al.,1994a), the GNRA tetraloop (Jucker & Pardi, 1995),23 S rRNA (Huang et al., 1996; Conn et al., 1999;Culver et al., 1999), U2 snRNA (Stallings & Moore,1997) and the HIV RNA (Puglisi & Puglisi, 1998).U-turns are stable structures that, as their nameimplies, induce a sharp change in the direction oftheir backbone. U-turns are one way to close hair-pin loops, but one of their most signi®cant proper-ties is their ability to create anchors for long-rangetertiary interactions due to the strong level ofexposure to solvent of the bases located 3Hto theturn. Probably the best example of this principle isutilized in protein synthesis to facilitate codon-anticodon base-pairing. The three nucleotidesof the anticodon are located immediately 3Hof a U-turn, rendering them accessible to long-rangecontacts with the codon and with the P site in 16 SrRNA (Prince et al., 1982; Cate et al., 1999). Themajority of the experimentally determined U-turnshave been associated with tertiary contacts. In thetRNA TcC-loop, the base located 3Hto the turnmakes a Watson-Crick pair with a guanosine basein the D-loop (G19:C56 in Yeast tRNAPhe), and thebase located immediately 5Hto the turn is involvedin the U54:A58 reverse Hoogsteen base-pair. Inseveral ribozymes, the bases following the G ofGNRA tetraloops are involved in a variety of long-range interactions (Jaeger et al., 1994; Pley et al.,1994b; Costa & Michel, 1995; Brown et al., 1996;Cate et al., 1996). Recently, U-turns have beeninferred in the formation of RNA/RNA inter-actions in natural antisense RNAs (Franch et al.,1999).The most salient structural feature of all U-turnmotifs is a sharp reversal of the RNA phosphodie-ster backbone, following a uridine base in the twotRNA U-turns (Quigley & Rich, 1976; Sussman &Kim, 1976), or a guanosine base in the GNRA U-turn (Jucker & Pardi, 1995). The turn is stabilizedby one or two hydrogen bonds forming betweenthe uridine or guanosine base that precedes theturn and the second base and phosphate followingthe turn. These stabilizing interactions are associ-ated with a set of sequence constraints that helpE-mail address of the corresponding author:robin.gutell@mail.utexas.edudoi:10.1006/jmbi.2000.3900 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 791±8030022-2836/00/040791±13 $35.00/0 # 2000 Academic Press
  2. 2. to identify them using comparative sequenceanalysis.Since U-turns are essential anchors for long-range interactions, their detection in large RNAmolecules such as rRNA could highlight signi®cantstructural elements in the folding and assembly ofthese complex entities. The goal here was to detectpotential U-turns in 16 S and 23 S rRNA. From thestructural characteristics of experimentally deter-mined U-turns, we de®ned a sequence/structuresignature for U-turns and sought instances thereofin our collection of comparative rRNA structuremodels. Potential U-turns in individual rRNAsequences were evaluated from a comparativestructural perspective. Those present in themajority of the rRNA sequences at homologouspositions were considered likely. The resultingU-turn candidates were classi®ed into tendistinct families, according to the predominantsequence (GNRA or UNR), loop type (hairpin,internal or multi-stem loop) and ¯anking basejuxtapositions (G:A, Y:N, etc.); candidates withtertiary interactions in proximity are consideredmore likely. Our previous (Gutell et al., 1994;Gutell, 1996) and current (see the CRW Website, http://www.rna.icmb.utexas.edu) covariationanalyses have identi®ed numerous tertiary inter-actions associated with potential U-turns.Results and DiscussionThe U-turn signatureFigure 1 is a schematic of tertiary interactions inseven types of U-turns for which a 3D crystal orNMR structure is available. Each nucleotide con-stituent is shown with a distinct geometrical ®gure(square, sugar; rectangle, base; circle, phosphate).Nucleotides are numbered starting at position 0 forthe uridine (or guanosine) preceding the turn, sothat positions following the turn are ‡1, ‡2, etc.The canonical U-turn motif involves two hydro-gen bonds, as they appear in the crystal structureof the Yeast tRNAPheanticodon loop (Figure 2,Westhof et al., 1988). The crucial interaction stabi-lizing the backbone reversal involves the uracilbase at position 0 and the phosphate groupimmediately following position 2. Although notabsolutely required in the anticodon function(Ashraf et al., 1999), this interaction is conserved inall known U-turn structures and replaced with aguanine-phosphate interaction in the GNRA loopU-turn (Jucker & Pardi, 1995).Another essential stabilizing hydrogen bond isbetween the uridine 2HOH at position 0 and thepurine N7 at position ‡2. An isosteric interactionoccurs in GNRA-type U-turns between the samepurine N7 and the 2Hhydroxyl of G0 (Jucker &Pardi, 1995). Purine bases are conserved at position‡2 in most of the U-turns studied (Figure 1),suggesting that this structure/sequence constraintshould be a component of the U-turn signature.The only exception to this rule is the anticodon U-turn, where position ‡2 is approximately evenlysplit between purines and pyrimidines in thetRNA sequence alignment (Sprinzl et al., 1991).Position ‡2 corresponds here to the central base ofthe anticodon, and is thus subjected to an aminoacid coding constraint that may con¯ict with thepurine constraint.Figure 1. Schematic of hydrogen bonds and base con-servation in several U-turn structures. U-turn-speci®cbase-base, base-sugar and base-phosphate H bonds areshown. The sequences shown (Y, pyrimidine; R, purine)are either essential for structure or conserved in homolo-gous molecules. (a) tRNA anticodon, 97 % consensussequence. (b) tRNA TÉC loop, 94 % consensus sequence.(c) Hammerhead U-turn, sequence required for ribo-zyme activity based on mutagenesis experiments(Ruffner et al., 1990). (d) 23 S rRNA 1082-1086, 70 % con-sensus sequence in Bacteria and chloroplasts, 89 % con-sensus in eukaryotes. (e) 23 S rRNA 1065-1073, 93 %consensus sequence in Bacteria and chloroplasts. (f)GUAANA loop, consensus based on three similar NMRand crystal structures, i.e. GUAAUA (Fountain et al.,1996; Huang et al., 1996), GUAACA (Stallings & Moore,1997) and GUAAAA (Puglisi & Puglisi, 1998). (g)GNRA loop, original consensus sequence observed inribosomal RNA alignments (Woese et al., 1990) andrequired for tertiary interactions (Heus & Pardi, 1991).792 U-turns in rRNA
  3. 3. A ®nal constraint on U-turns has recently beenrevealed in a study of the anticodon U-turn(Auf®nger & Westhof, 1999). The authors observedthat the ®rst and last nucleotides of the anticodonloop (positions 32:38, with sequences C:A, U:A,U:U, C:C or U:C, see Figure 2) form a non-canoni-cal base-pair that acts as an interface between theend of the anticodon stem (base-pair 31:39) and theU-turn at position 33. The hammerhead ribozymealso features a Y:Y base-pair 5Hof a U-turn (Pleyet al., 1994a), although its conformation differsfrom that of the tRNA 32:38 pair. Indeed, all U-turns in Figure 1 contain a non-canonical pair atthe 5Hside of the turn.An inspection of the anticodon U-turn in Figure 2provides a possible explanation for the absence ofWatson-Crick pairs ¯anking the U-turn. The non-canonical C32:A38 base-pair is shown in orangewhile a hypothetical guanosine base (red) is posi-tioned to form a Watson-Crick pair with C32. Inthis scenario, the displacement of the C1Hatomsbetween this guanosine base and A38 is about9.3 AÊ . Here, the rotation of this guanosine base isinadequate to connect properly to residue A36without disrupting the whole loop conformation(Auf®nger & Westhof, 1999). Intercalation of extraresidues between A36 and the hypothetical guano-sine is also not possible.There is an example where the UGA sequencemotif does not form a U-turn because the ¯ankingnucleotides form a normal Watson-Crick base-pair.The solution structure for the 5H-GGUG[UGAA]-CACC oligonucleotide, representative of thetetraloop positions 1516-1519 in 16 S rRNA, doesnot make a U-turn (Butcher et al., 1997). The basejuxtapositions ¯anking most U-turns are Y:H(H ˆ A, C or U), except in GUAANA loops(Figure 1(f)), where it is a sheared G:A pair(Fountain et al., 1996; Huang et al., 1996; Stallings& Moore, 1997; Puglisi & Puglisi, 1998). Therefore,we favor U-turn candidates ¯anked by Y:H or G:Abase juxtapositions, although the conformation forthese non-canonical pairs is not the same.The U-turn signature emerging from this anal-ysis is presented in Figure 3. This sequence motifdoes not include the GNRA-type U-turn, for whichthe GNRA sequence requirement is well estab-lished (Woese et al., 1990; Heus & Pardi, 1991). TheUNR-type U-turn typically features a conservedFigure 2. Stereo representation of the Yeast tRNAPheanticodon loop (Westhof et al., 1988), from A31 to U39. Resi-due 37 has been omitted for clarity. The anticodon is blue and the closing base-pair is purple. The turning uridinebase (33) is black. Hydrogen bonds between U3-3 and A36 stabilizing the U-turn are shown with broken lines. Thenon-canonical 32:38 base-pair ¯anking the U-turn is orange, with its bifurcated hydrogen bond (Auf®nger & Westhof,1999) shown with a broken line. A hypothetical guanosine base (red) has been positioned to form a Watson-Crickpair with C32, showing the effect of a canonical base-pair at this position. The C1Hatom of this hypothetical guano-sine base is displaced by 9.26 AÊ from the C1Hof A38. Exposed H bond donors and acceptors in the three bases andsugars following the turn are shown with ``hard spheres.Figure 3. Consensus sequence and structure for UNR-type U-turns.U-turns in rRNA 793
  4. 4. Table 1. U-turn candidates in 16 S and 23 S rRNACategory rRNA LT LC UP TCA. Canonical GNRA hairpin loops 16 S H 159-162 1 None16 S H 297-300 1 None16 S H 727-730 1 None16 S H 898-901 1 None16 S H 1077-1080 1 None16 S H 1266-1269 1 None23 S H 463-466 1 None23 S H 630-633 1 None23 S H 1223-1226 1 None23 S H 2375-2378 1 None23 S H 2595-2598 1 None23 S H 2659-2662 1 ‡2: [2661(2550:2558)]23 S H 2857-2860 1 NoneB. GNRA in larger hairpin loops 16 S H 1315-1322 2 None23 S H 306-311 2 None23 S H 745-752 4 À2: [746(2057:2611)]23 S H 780-784 1 NoneC. GNRA in internal and multi-stem loops16 S M 765-768 1 None16 S M 1108-1112 1 ‡1: [1109(933:1384)]23 S M 215-223 6 À1: [219(234:430)];‡1: [221(265:427)]23 S M 475-483 2 None23 S I 511-515 2 None23 S M 818-821 1 None23 S I 1565-1572 4 None23 S M 1668-1681 7 NoneD. UNR in trinucleotide hairpinloops23 S H 1083-1085 1 ‡1: [1084(1054:1105)];‡2: [1085(1055:1104)]23 S H 1926-1928 1 ‡2: (1834:1928)E. UNR at position 2 flanked byY:R or Y:Y base-pairs16 S I 13-16 2 À1: (13:920); 0: (14:1398);‡1: (15:1397); ‡2: (16:920)16 S H 322-331 2 None16 S H 618-622 2 None16 S H 1090-1095 2 None23 S H 567-574 2 ‡6: (574:2034)23 S H 1065-1073 2 ‡5: [1071(1091:1100)];‡6: [1072(1092:1099)]F. UNR in hairpin loops flankedby G:A base-pairs16 S H 260-266 2 None16 S H 691-696 2 None23 S H 714-717 1 None23 S H 1093-1098 2 None
  5. 5. G.UNRininternalandmutli-stemloopsflankedbyC:Gbase-pairs16SM1211-12141None23SI1352-13541None23SM2197-21991NoneH.UNRinloopsflankedbyotherbase-pairs16SH787-7953None16SM1065-10671None16SH1358-13641None23SH1951-19551‡3:[1954(1950:1956)]23SI2738-27412NoneI.UNRinloopswithoutflankingbase-pairs16SM116-12130:(118:288);‡1:(119:287);‡3:[121(124:237)(125:236)]16SM557-5665‡5:(566:919)23SM446-4603None23SM1339-13422‡3:(1343:1404);‡4:(1344:1403)23SI2162-21736‡2:(2112:2169);‡3:(2113:2170);‡5:(2117:2172)23SH2265-227580:(2272:2564)23SM2422-24332À2:[(2420:2396)2180];‡4:(2427:2282);‡5:(2428:2359)23SM2448-24542‡4:(2453:2499);‡5:(2454:2498)23SM2497-25064À2:(2498:2454);À1:(2499:2453);‡4:(2504:2447)and[(2504:2447)2508]23SM2583-25873‡1:(2586:1782)J.AmbigousUNR/GNRAexchanges16SH863-86610:[863(570:866)];‡2:(865:571);‡3:(866:570)16SH1013-101610,‡3:(1013:1016)AllpositionnumberingisdoneusingtheE.colistructuremodelreferencesequencenumberingsystem.LT,Looptype,whereH,hairpin;I,internal;andM,multi-stem.LC,Loopcoordinates;indicatingtherangeofnucleotidesincludedintheloop.UP,U-turnposition,wherethe®rstnucleotideoftheloopˆ1.TC,Comparativestructuremodeltertiarycoordinates,intheformat(posi-tionrelativetoturn):(tertiary).Tertiaryinteractionsoccuringinthefavoredregion(nucleotidesÀ1through‡3)areinbold.Tentativetertiaryinteractionsareinitalics.Fulldetailsofthecompara-tivestructuremodelsareavailableathttp://www.rna.icmb.utexas.edu/ANALYSIS/U-TURN/.
  6. 6. purine base at position ‡2 (a constraint that doesnot apply to the anticodon U-turn) and a ¯ankingY:A, Y:Y or G:A pair at position À1 (this positionshould not be occupied by a canonical Watson-Crick base-pair). The base-pairing partner of pos-ition À1 does not necessarily belong to the sameloop as the U-turn and can be a distant nucleotide(e.g. in the 16 S rRNA 1211 motif, the ¯ankingbase-pair is 1047:1210, see Figure 1). Tertiarycontacts at positions À1, ‡1, ‡2, and ‡3 will beinvestigated.GNRA patternsConserved GNRA patterns occur at 25 positionsin the 16 S and 23 S rRNAs. We de®ne here ``con-served as present in a minimum of 80 % of thebacterial sequences. GNRA patterns are foundunder the following forms.Canonical GNRA hairpin loopsThe canonical form of the GNRA U-turn is thefour-nucleotide hairpin loop, with 13 occurrencesoverall (Figure 4 and Table 1, category A). Some ofthese candidates have been con®rmed experimen-tally, including the 23 S rRNA 2659:2662 tetraloopat the tip of the sarcin-ricin loop (Szewczak &Moore, 1995; Correll et al., 1998). Comparativeanalysis of this loop suggests a base-tripleinteraction between position ‡2 (2661) andthe base-pair 2550:2558 (see the CRW site,http://www.rna.icmb.utexas.edu). Whereas thisbase-triple covariation is not consistent with theloop-loop interaction observed in the recent 50 Ssubunit crystal structure (Ban et al., 1999),rearrangements remain possible and should beconsidered in future studies of rRNA dynamics.The recent low-resolution crystal structure of the70 S ribosome also suggests an interaction betweenthe canonical GNRA loop at position 16 S:898-901and the 790 helix of 23 S rRNA (Cate et al., 1999).Two tetraloops with GNRA/GNRG sequence vari-ations were also included (16 S:727-730 and23 S:630-633), since both sequences can fold in thesame way (Murphy & Cech, 1994). This is con-®rmed for the 16 S rRNA loop 727-730 in theS15,S6,S18 rRNA crystal structure (Agalarov et al.,2000).GNRA in larger hairpin loopsFour conserved GNRA motifs occur within hair-pin loops that contain more than four nucleotides(Table 1 and Figure 4, category B). While one ofthese (23 S:306) is located within a 6 nt loop and is¯anked on both sides with nucleotides that canform a non-canonical base-pair, the GNRAsequence is placed asymmetrically in the otherhairpin loops. In these cases, the loop needs to bedistorted to accommodate a GNRA structure.Although this is theoretically possible, there is noexperimental precedent. The example at positions780-784 in 23 S rRNA would have a bulgedK (G or U) following the GNRA structure, whilethe two remaining GNRA motifs occur in loops ofsize eight, at the second or fourth loop position.GNRA in internal and multi-stem loopsEight conserved GNRA sequences occur ininternal or multi-stem loops (Figure 4 and Table 1,category C). GNRA tetraloop conformations havenever been observed experimentally in such situ-ations; therefore, these should be considered tenta-tive. The GNRA sequence is involved in a putativetertiary interaction in at least one of these loops (seethe CRW site, http://www.rna.icmb.utexas.edu).The strongest example occurs in the 1108 loop of16 S rRNA, where position 1109 (‡1) covaries withthe 933:1384 base-pair, while in the 215 loop of 23 SrRNA, base triple covariations occur at positions ‡1and À1. Two other internal loops display signi®cantlevels of GNRA/GNRG variation: 16 S rRNA765-768 (3.4 % GAAG) and 23 S rRNA 818-821(33 % GAAG) (see above).UNR patternsUNR patterns conserved in more than 90 % ofthe bacterial sequences are found at 44 sites in 16 Sand 23 S rRNA. We eliminate 11 of these sites thatare ¯anked by Watson-Crick pairs with multiplecompensatory base changes, since we do notexpect U-turns to be enclosed by standard base-pairs. The remaining 33 sites are ¯anked byunpaired nucleotides or by a highly conservedWatson-Crick base juxtaposition (e.g. 95 % U:A)that could possibly form a non-canonical base-pair.While the majority of candidates occur in hairpinor multi-stem loops, where UNR-type U-turnshave already been observed experimentally, threeoccur in internal loops (Table 1), an unexpectedand structurally less likely situation. Candidates ofthe UNR type were classi®ed into categories Dthrough J (Table 1).UNR in trinucleotides hairpin loopsTwo trinucleotide hairpin loops contain the UNRmotif directly closed by a single base-pair which ishighly conserved (Figure 4 and Table 1, categoryD). The 23 S rRNA base-pair 1082:1086 is U:A innearly 100 % of the bacterial and chloroplastsequences and C:G in almost all of the eukaryoticsequences (Table 2). Such an atypical base-pairingconstraint can be associated to various confor-mations of non-canonical base-pairs (Gautheret &Gutell, 1997). Indeed, a reverse Watson-Crick base-pair at position 1082:1086 and a U-turn in theUAA hairpin were identi®ed in the crystal struc-ture of the L11 binding region of 23 S rRNA (Connet al., 1999; Wimberly et al., 1999). The 1926 triloopalso has the UAA sequence; here, the closing base-pair is a conserved C:G in Bacteria and chloro-plasts (Table 2). While reverse Watson-Crick U:A796 U-turns in rRNA
  7. 7. and C:G base-pairs do not form identical isostericconformations (Gautheret & Gutell, 1997), a pre-cedent for this type of exchange is the tRNA 15:48reverse Watson-Crick base-pair. Given similarsequence constraints in both loops, we expect their3D structure to be similar as well. Since the bases‡1 and ‡2 in the 23 S rRNA 1083-1085 loop areinvolved in tertiary contacts with base-pairs1054:1105 and 1055:1104 in the crystal structure ofthe L11 binding region of 23 S rRNA (Conn et al.,1999), we anticipated tertiary interactions at pos-itions 1927 or 1928. Interestingly, our comparativeanalysis revealed a covariation between positions1928 and 1834 (see Figure 4 and the CRW site,http://www.rna.icmb.utexas.edu/).UNR at position 2 flanked by Y:R orY:Y base-pairsSix UNR sites have the UNR pattern at position2 of a hairpin or internal loop, and ¯anked by aY:R or Y:Y juxtaposition (Figure 4 and Table 1, cat-egory E). This arrangement is similar to the tRNAanticodon U-turn, except for the difference in loopsizes. Tertiary contacts at position ‡1 to ‡3 havebeen predicted for loop 16 S:13 (see the CRW site,http://www.rna.icmb.utexas.edu/), with covaria-tions at positions 13:920 (À1), 14:1398 (U-turnposition), 15:1397 (‡1) and 16:920 (‡2). Althoughthe U-turn position has not been implicated inlong-range tertiary interactions, contacts withposition À1 are possible, as shown in the hammer-head ribozyme (Pley et al., 1994a) and TcC-loop oftRNA (Quigley & Rich, 1976). In addition, two ofthe U-turn candidates in this category have tertiaryinteractions at positions 5 and 6 (23 S rRNAposition 567 and 1065, see Table 1 and Figure 4).UNR in hairpin loops flanked by G:A base-pairsFour UNR motifs are ¯anked by a G:A base jux-taposition (Figure 4 and Table 1, category F). Hexa-nucleotide loops with the GUAANA sequenceconsensus fall in this category, forming a wellcharacterized three-dimensional motif (Fountainet al., 1996; Huang et al., 1996; Stallings & Moore,1997; Puglisi & Puglisi, 1998), with a sheared G:Aclosing base-pair and a U-turn forming at the con-served uridine base. Nucleotides AAU located 3Htothe U-turn in the 23 S rRNA 1093 loop form ter-tiary contacts with the 1065-1073 loop (Conn et al.,1999). Likewise, the 23 S rRNA 713 loop isinvolved in an important tertiary interaction brid-ging the 30 S and 50 S ribosomal subunits (Culveret al., 1999). The 16 S rRNA hairpin loop 691-696begins with a G and ends with an A and is similarto the previous two motifs in size and loop closure.Interestingly, this loop is protected by tRNA(Moazed & Noller, 1989b) and the association ofsubunits (Powers et al., 1993; Merryman et al.,1999). The fourth motif, 16 S rRNA 260-266, has aseven-nucleotide loop with a weak covariation inthe Bacteria between positions 260 and 265. Thiswould create a ®ve-nucleotide hairpin loop with aG:G, G:A or A:A closing base-pair. These pairingtypes can adopt a sheared base-pair conformationsimilar to the G:A pair in the other motif.Sequence variations in the 23 S rRNA 713 loopare particularly interesting (Table 3). Archaea andeukaryotes have a central GAAA sequence closedby a Watson-Crick base-pair (G:C or C:G), whileBacteria and chloroplasts have a central UNANsequence closed by a G:A juxtaposition. Both com-binations (Watson-Crick pair ‡ GNRA sequence orG:A pair ‡ UNRN sequence) can form a U-turn atposition 2 of the loop, and thus retain the ability toform the tertiary interaction with the 30 S riboso-mal subunit.UNR in internal and multi-stem loops flanked byC:G pairsThree internal and multi-stem loops display con-served UNA sequences adjacent to a conservedC:G pair in Bacteria (see Figure 4 and Table 1, cat-egory G, and the CRW site for base-pair frequen-cies). It is unlikely that reverse Watson-Crick C:Gpairs form in these cases, since the pairs are¯anked by other secondary structure base-pairs.Although other non-canonical conformations arestill possible, these three sites are weak U-turn can-didates. An additional site in this category, foundat 23 S rRNA position 202, has been eliminated,since it is part of a ``loop E motif (Leontis &Westhof, 1998), which does not contain a U-turn.Table 2. Base-pair frequencies for 23 S rRNA positions1082:1086 and 1925:1929 (only frequencies over 1 % areshown)Kingdom Most frequent sequenceA. 1082:1086(eu)Bacteria U:A (98.8 %) C:G (1.0 %)(1 phylogenetic event)aChloroplast U:A (98.0 %) U:C (2.0 %)Archaea C:G (59.5 %) U:A (40.5 %)(3 phylogenetic events)aEucarya C:G (98.6 %)B. 1925:1929(eu)Bacteria C:G (99.2 %)Chloroplast C:G (100.0 %)Archaea U:G (100.0 %)Eucarya C:G (99.4 %)aConcerted base changes occurring between closely relatedorganisms (see Materials and Methods).Table 3. Sequence variations at 23 S rRNA positions713-718Kingdom Most frequent sequence(eu)Bacteria GUAANA (96 %)Chloroplast GUNANA (85 %)Archaea CGAAAG (40 %) GGAAAC(35 %) CUUACG (8 %)Eucarya GGAAAC (80 %) CGAAAG(5 %)U-turns in rRNA 797
  8. 8. UNR in loops flanked by other base-pairsFive UNR sequences are ¯anked by other basejuxtapositions (Figure 4 and Table 1, category H).Two of these are ¯anked by a secondary structurebase-pair (23 S:1951 and 16 S:1065), but atypicalsequence constraints in these pairs are compatiblewith a non-canonical pairing (Table 4). In addition,the 23 S rRNA 1951 U-turn candidate is associatedto a base-triple type covariation between positions(1950:1956) and 1954 (Figure 4 and the CRWsite, http://www.rna.icmb.utexas.edu/). The 16 SFigure 4 (legend shown on page 800)798 U-turns in rRNA
  9. 9. rRNA 787-795 loop contains two overlapping U-turn signatures: the 95 % consensus for 788-790 isUYA, while the 83 % consensus for 789-791 isUAG. The structure of this nine-nucleotide hairpinresembles the tRNA TcC-loop (Gu et al., 1994),with a closing A:C base-pair reducing the loop sizeto seven. The U-turn occurring at position U55 intRNA would be homologous to position U789 in16 S rRNA (Gu et al., 1994). In addition, in vitroselection experiments indicate that U789, ratherthan U788 is required for ribosome function (Leeet al., 1997). Therefore, while sequence conservationalone would favor a U-turn at 788, this turn ismore likely at position 789. Nucleotides within thisFigure 4 (legend shown on page 800)U-turns in rRNA 799
  10. 10. Figure 4. Potential U-turns shown on the E. coli secondary structures for the small subunit (a), the large subunit 5Hhalf (b) and the large subunit 3Hhalf (c) of ribosomal RNA. Loops containing potential U-turns are shown as nucleo-tides, and the remainder of the structure is shown as gray circles. Each U-turn position is shown as a red nucleotide.Green nucleotides show positions involved in tertiary interactions. U-turn positions involved in tertiary interactionshave red nucleotides enclosed in green boxes. Tentatively proposed interactions in proximity to U-turns are blue. Yel-low boxes highlight hairpin loops and their loop type identi®ers; orange boxes highlight internal and multi-stemloops and their loop type identi®ers. U-turn categories are de®ned as in Table 1: A, canonical GNRA hairpin loops;B, GNRA in larger hairpin loops; C, GNRA in internal and multi-stem loops; D, UNR in trinucleotide hairpinloops; E, UNR at position 2 ¯anked by Y:R or Y:Y base-pairs;, UNR in internal and multi-stem loops ¯ankedby C:G base-pairs; H, UNR in loops ¯anked by other base-pairs; I, UNR in loops without ¯anking base-pairs;J, ambiguous UNR/GNRA exchanges.800 U-turns in rRNA
  11. 11. loop are protected by ribosomal subunit associ-ation, suggesting that this U-turn motif is involvedin tertiary interactions (Powers et al., 1993;Merryman et al., 1999).UNR in loops without flanking base-pairFlanking base-pairs are unknown or ambiguousfor ten of the U-turn candidates (Figure 4 andTable 1, category I). Four of these potential U-turnsare associated with predicted tertiary interactionsat position ‡1 to ‡3 relative to the turn (summar-ized in Table 1). The 116 loop in 16 S rRNA con-tains two predicted interactions: a base triple atpositions 121(124:237) or 121(125:236) (Babin et al.,1999) and the two base-pairs 118:288 and 119:287(see putative tertiary interactions on the CRW site).The latter interaction is supported by U.V. cross-linking (Stiege et al., 1986). Proposed tertiary inter-actions at positions 2112:2169 and 2113:2170(Figure 4 and the CRW site) in the rRNA E site(23 S rRNA 2162 loop) (Moazed & Noller, 1989a)are also supported by crosslinking studies (Doringet al., 1991). These tertiary interactions correspondto positions ‡2 and ‡3 after the proposed U-turn.Tertiary base covariations are also observed at the3Hend of the 23 S rRNA 1339 loop (pseudoknot1343-1344:1403-1404, see the CRW cite) and in the23 S rRNA 2583 loop (1782:2586). Both of theseproposed interactions are supported experimen-tally, the former by site-directed mutagenesis (Kooiet al., 1993), and the latter by U.V. crosslinksbetween positions 2584-2588 and 1777-1792 (Stiegeet al., 1983). The 23 S:2497 loop has putativetertiary interactions 5Hto the turn at positions À1and À2 (2499:2453 and 2498:2454).Ambiguous UNR/GNRA exchangesExchanges between UNR and GNRA sequencesoccur at two hairpin loop sites (16 S:863 and16 S:1013, see Figure 4). This is similar to thesequence variation at the 23 S:713 hairpin loop(Table 3). This type of variation could result from aselective pressure for U-turns at these sites. The16 S:863 loop has another characteristic associatedwith U-turns: two positions 3Hto this putative U-turn form tertiary pseudoknot base-pairs to 16 SrRNA positions 570-571 (Gutell et al., 1986; Vilaet al., 1994). However, in both cases the UNRsequence is not ¯anked by a G:A or Y:H mismatch,but instead by canonical base-pair exchanges (e.g.G:C to A:U). The structures for these two sites areuncertain, since these pairing types have not beenobserved ¯anking a U-turn.ConclusionComparative sequence analysis enables us to dis-tinguish randomly occurring U-turn signaturesfrom candidates that are supported by sequenceconservation and speci®c patterns of base-pairexchanges. We have identi®ed 58 UNR and GNRAU-turn candidates in a variety of structural settingsin the 16 S and 23 S rRNAs. Since the sequenceand structural information that de®nes a U-turn isminimal and the sequence constraint rules that wehave used to identify U-turns may be associatedwith other structural motifs, some of our predictedU-turns may be incorrect. Alternatively, these U-turn signatures may be associated with structuralconformations that alternate between U-turns andthese other structural motifs. Used as workinghypotheses, putative U-turns and the associatedtertiary interactions can be used for modeling(prior to re®nement) and interpretation (afterre®nement) of the X-ray crystal structures of theribosome.Materials and MethodsWe have used the alignments of small and large sub-unit rRNA sequences maintained by us at the Universityof Texas (R.R.G., unpublished results). The small subunitrRNA alignment contains 5826 Bacteria, 182 chloroplast,264 Archaea and 1054 Eukaryotic sequences. The largesubunit rRNA alignment contains 326 Bacteria, 103chloroplast, 41 Archaea and 263 Eukaryotic sequences.Secondary structure diagrams for representatives of themain phylogenetic groupings are inferred with compara-tive sequence analysis (Gutell et al., 1993; Gutell, 1994)and are available from our Austin, Texas CRW site (TheComparative RNA Web Site: http://www.rna.icmb.u-texas.edu/, R.R.G., unpublished results).Base frequencies were computed independently in theBacteria, chloroplast, Archaea and Eukaryotic align-ments. When not otherwise speci®ed, base or base-pairfrequencies refer only to Bacteria sequences. Base num-bering always refers to Escherichia coli 16 S or 23 S rRNAsequences (GeneBank accession no. J01695). The phyloge-netic events for base-pairs in Tables 2 and 4 werederived from the CRW site (http://www.rna.icmb.utexas.edu/). Here, the numbers of mutual changes thathave occurred throughout evolution for each pair in ourcomparative structure model are accessible, as well asTable 4. Sequence variations at base-paired positions16 S rRNA 1064:1192 and 23 S rRNA 1950:1956 (allfrequencies over 1 % are shown, see the CRW web sitefor a detailed analysis)Kingdom Most frequent sequenceA. 16 S 1064:1192(eu)Bacteria G:C (97 %) G:U (2 %)Archaea G:C (100 %)Eucarya C:U (89 %) U:C (4 %) U:A (2 %)(4 phylogenetic events)aB. 23 S 1950:1956(eu)Bacteria G:U (94 %) U:A (5 %)(2 phylogenetic events)aArchaea G:U (59 %) U:G (17 %) A:A(15 %) U:A (10 %) (nophylogenetic event)aEucarya C:A (89 %) G:U (3 %) U:G (3 %)U:U (3 %) (2 phylogeneticevents)aaConcerted base changes occurring between closely relatedorganisms (see Materials and Methods).U-turns in rRNA 801
  12. 12. details of the base-pair types and speci®c phylogeneticlocation for each mutual change. A ``phylogenetic eventwas recorded when both positions in the pair variedbetween two consecutive organisms. This approximationis simplistic but conservative, since all but the mostrecent events are neglected.tRNA base frequencies, were derived from the 1997version of M. Sprinzls tRNA alignments (Sprinzl et al.,1991). All nuclear tRNAs and tDNAs were included inour base counts. The Yeast tRNAPhenumbering is usedthroughout.The Figures and Tables for this article are availableonline at the main CRW site (http://www.rna.icmb.u-texas.edu/, go to ``RNA Structure Analysis/U-Turn) orby using the speci®c URL (http://www.rna.icmb.utexa-s.edu/ANALYSIS/U-TURN/).AcknowledgmentsThis work was supported in part from the NIH grantsawarded to R.G. (NIH - GM48207) and startup fundsfrom the Institute for Cellular and Molecular Biology atthe University of Texas at Austin.ReferencesAgalarov, S. 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