1. Predicting U-turns in Ribosomal RNA with
Comparative Sequence Analysis
Robin R. Gutell1
*, Jamie J. Cannone1
, Danielle Konings2
and Daniel Gautheret3
1
Institute for Cellular and
Molecular Biology, University
of Texas at Austin, 2500
Speedway, Austin, TX 78712-
1095, USA
2
Department of Molecular
Cellular and Developmental
Biology, University of Colorado
Campus Box 347, Boulder
CO 80309-0347, USA
3
Structural and Genetic
Information, CNRS UMR
1889, 31 chemin Joseph
Aiguier, 13 402, Marseille
Cedex 20, France
The U-turn is a well-known RNA motif characterized by a sharp reversal
of the RNA backbone following a single-stranded uridine base. In exper-
imentally determined U-turn motifs, the nucleotides 3H
to the turn are fre-
quently involved in tertiary interactions, rendering this motif particularly
attractive in RNA modeling and functional studies. The U-turn signature
is 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 set
of aligned 16 S and 23 S rRNA sequences. U-turn candidates occur in
hairpin loops (34 times) as well as in internal and multi-stem loops (24
times). These are classi®ed into ten families based on loop type, sequence
pattern (UNR or GNRA) and the nature of the closing base juxtaposition.
In 13 cases, the bases on the 3H
side of the turn, or on the immediate 5H
side, are involved in tertiary covariations, making these sites strong can-
didates for tertiary interactions.
# 2000 Academic Press
Keywords: ribosomal RNA; comparative sequence analysis; U-turns;
tertiary interactions*Corresponding author
Introduction
U-turns are small RNA structural motifs that
were ®rst discovered in the anticodon and TcC-
loop of tRNA (Quigley & Rich, 1976) and later
identi®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 name
implies, induce a sharp change in the direction of
their 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-range
tertiary interactions due to the strong level of
exposure to solvent of the bases located 3H
to the
turn. Probably the best example of this principle is
utilized in protein synthesis to facilitate codon-
anticodon base-pairing. The three nucleotides
of the anticodon are located immediately 3H
of a U-
turn, rendering them accessible to long-range
contacts with the codon and with the P site in 16 S
rRNA (Prince et al., 1982; Cate et al., 1999). The
majority of the experimentally determined U-turns
have been associated with tertiary contacts. In the
tRNA TcC-loop, the base located 3H
to the turn
makes a Watson-Crick pair with a guanosine base
in the D-loop (G19:C56 in Yeast tRNAPhe
), and the
base located immediately 5H
to the turn is involved
in the U54:A58 reverse Hoogsteen base-pair. In
several ribozymes, the bases following the G of
GNRA 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 been
inferred in the formation of RNA/RNA inter-
actions in natural antisense RNAs (Franch et al.,
1999).
The most salient structural feature of all U-turn
motifs is a sharp reversal of the RNA phosphodie-
ster backbone, following a uridine base in the two
tRNA U-turns (Quigley & Rich, 1976; Sussman &
Kim, 1976), or a guanosine base in the GNRA U-
turn (Jucker & Pardi, 1995). The turn is stabilized
by one or two hydrogen bonds forming between
the uridine or guanosine base that precedes the
turn and the second base and phosphate following
the turn. These stabilizing interactions are associ-
ated with a set of sequence constraints that help
E-mail address of the corresponding author:
robin.gutell@mail.utexas.edu
doi:10.1006/jmbi.2000.3900 available online at http://www.idealibrary.com on J. Mol. Biol. (2000) 300, 791±803
0022-2836/00/040791±13 $35.00/0 # 2000 Academic Press

2. to identify them using comparative sequence
analysis.
Since U-turns are essential anchors for long-
range interactions, their detection in large RNA
molecules such as rRNA could highlight signi®cant
structural elements in the folding and assembly of
these complex entities. The goal here was to detect
potential U-turns in 16 S and 23 S rRNA. From the
structural characteristics of experimentally deter-
mined U-turns, we de®ned a sequence/structure
signature for U-turns and sought instances thereof
in our collection of comparative rRNA structure
models. Potential U-turns in individual rRNA
sequences were evaluated from a comparative
structural perspective. Those present in the
majority of the rRNA sequences at homologous
positions were considered likely. The resulting
U-turn candidates were classi®ed into ten
distinct families, according to the predominant
sequence (GNRA or UNR), loop type (hairpin,
internal or multi-stem loop) and ¯anking base
juxtapositions (G:A, Y:N, etc.); candidates with
tertiary interactions in proximity are considered
more likely. Our previous (Gutell et al., 1994;
Gutell, 1996) and current (see the CRW Web
site, http://www.rna.icmb.utexas.edu) covariation
analyses have identi®ed numerous tertiary inter-
actions associated with potential U-turns.
Results and Discussion
The U-turn signature
Figure 1 is a schematic of tertiary interactions in
seven types of U-turns for which a 3D crystal or
NMR 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 for
the uridine (or guanosine) preceding the turn, so
that positions following the turn are ‡1, ‡2, etc.
The canonical U-turn motif involves two hydro-
gen bonds, as they appear in the crystal structure
of the Yeast tRNAPhe
anticodon loop (Figure 2,
Westhof et al., 1988). The crucial interaction stabi-
lizing the backbone reversal involves the uracil
base at position 0 and the phosphate group
immediately following position 2. Although not
absolutely required in the anticodon function
(Ashraf et al., 1999), this interaction is conserved in
all known U-turn structures and replaced with a
guanine-phosphate interaction in the GNRA loop
U-turn (Jucker & Pardi, 1995).
Another essential stabilizing hydrogen bond is
between the uridine 2H
OH at position 0 and the
purine N7 at position ‡2. An isosteric interaction
occurs in GNRA-type U-turns between the same
purine N7 and the 2H
hydroxyl 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 constraint
should be a component of the U-turn signature.
The only exception to this rule is the anticodon U-
turn, where position ‡2 is approximately evenly
split between purines and pyrimidines in the
tRNA sequence alignment (Sprinzl et al., 1991).
Position ‡2 corresponds here to the central base of
the anticodon, and is thus subjected to an amino
acid coding constraint that may con¯ict with the
purine constraint.
Figure 1. Schematic of hydrogen bonds and base con-
servation in several U-turn structures. U-turn-speci®c
base-base, base-sugar and base-phosphate H bonds are
shown. The sequences shown (Y, pyrimidine; R, purine)
are either essential for structure or conserved in homolo-
gous molecules. (a) tRNA anticodon, 97 % consensus
sequence. (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 NMR
and 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 in
ribosomal RNA alignments (Woese et al., 1990) and
required for tertiary interactions (Heus & Pardi, 1991).
792 U-turns in rRNA

3. A ®nal constraint on U-turns has recently been
revealed in a study of the anticodon U-turn
(Auf®nger & Westhof, 1999). The authors observed
that the ®rst and last nucleotides of the anticodon
loop (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 the
end of the anticodon stem (base-pair 31:39) and the
U-turn at position 33. The hammerhead ribozyme
also features a Y:Y base-pair 5H
of a U-turn (Pley
et al., 1994a), although its conformation differs
from that of the tRNA 32:38 pair. Indeed, all U-
turns in Figure 1 contain a non-canonical pair at
the 5H
side of the turn.
An inspection of the anticodon U-turn in Figure 2
provides a possible explanation for the absence of
Watson-Crick pairs ¯anking the U-turn. The non-
canonical C32:A38 base-pair is shown in orange
while a hypothetical guanosine base (red) is posi-
tioned to form a Watson-Crick pair with C32. In
this scenario, the displacement of the C1H
atoms
between this guanosine base and A38 is about
9.3 AÊ . Here, the rotation of this guanosine base is
inadequate to connect properly to residue A36
without disrupting the whole loop conformation
(Auf®nger & Westhof, 1999). Intercalation of extra
residues between A36 and the hypothetical guano-
sine is also not possible.
There is an example where the UGA sequence
motif does not form a U-turn because the ¯anking
nucleotides form a normal Watson-Crick base-pair.
The solution structure for the 5H
-GGUG[UGAA]-
CACC oligonucleotide, representative of the
tetraloop positions 1516-1519 in 16 S rRNA, does
not make a U-turn (Butcher et al., 1997). The base
juxtapositions ¯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:A
base juxtapositions, although the conformation for
these non-canonical pairs is not the same.
The U-turn signature emerging from this anal-
ysis is presented in Figure 3. This sequence motif
does not include the GNRA-type U-turn, for which
the GNRA sequence requirement is well estab-
lished (Woese et al., 1990; Heus & Pardi, 1991). The
UNR-type U-turn typically features a conserved
Figure 2. Stereo representation of the Yeast tRNAPhe
anticodon 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 uridine
base (33) is black. Hydrogen bonds between U3-3 and A36 stabilizing the U-turn are shown with broken lines. The
non-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-Crick
pair with C32, showing the effect of a canonical base-pair at this position. The C1H
atom of this hypothetical guano-
sine base is displaced by 9.26 AÊ from the C1H
of A38. Exposed H bond donors and acceptors in the three bases and
sugars 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. Table 1. U-turn candidates in 16 S and 23 S rRNA
Category rRNA LT LC UP TC
A. Canonical GNRA hairpin loops 16 S H 159-162 1 None
16 S H 297-300 1 None
16 S H 727-730 1 None
16 S H 898-901 1 None
16 S H 1077-1080 1 None
16 S H 1266-1269 1 None
23 S H 463-466 1 None
23 S H 630-633 1 None
23 S H 1223-1226 1 None
23 S H 2375-2378 1 None
23 S H 2595-2598 1 None
23 S H 2659-2662 1 ‡2: [2661(2550:2558)]
23 S H 2857-2860 1 None
B. GNRA in larger hairpin loops 16 S H 1315-1322 2 None
23 S H 306-311 2 None
23 S H 745-752 4 À2: [746(2057:2611)]
23 S H 780-784 1 None
C. GNRA in internal and multi-
stem loops
16 S M 765-768 1 None
16 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 None
23 S I 511-515 2 None
23 S M 818-821 1 None
23 S I 1565-1572 4 None
23 S M 1668-1681 7 None
D. UNR in trinucleotide hairpin
loops
23 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 by
Y:R or Y:Y base-pairs
16 S I 13-16 2 À1: (13:920); 0: (14:1398);
‡1: (15:1397); ‡2: (16:920)
16 S H 322-331 2 None
16 S H 618-622 2 None
16 S H 1090-1095 2 None
23 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 flanked
by G:A base-pairs
16 S H 260-266 2 None
16 S H 691-696 2 None
23 S H 714-717 1 None
23 S H 1093-1098 2 None

5. G.UNRininternalandmutli-
stemloopsflankedbyC:Gbase-
pairs
16SM1211-12141None
23SI1352-13541None
23SM2197-21991None
H.UNRinloopsflankedbyother
base-pairs
16SH787-7953None
16SM1065-10671None
16SH1358-13641None
23SH1951-19551‡3:[1954(1950:1956)]
23SI2738-27412None
I.UNRinloopswithoutflanking
base-pairs
16SM116-12130:(118:288);‡1:(119:287);
‡3:[121(124:237)(125:236)]
16SM557-5665‡5:(566:919)
23SM446-4603None
23SM1339-13422‡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/GNRA
exchanges
16SH863-86610:[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. purine base at position ‡2 (a constraint that does
not apply to the anticodon U-turn) and a ¯anking
Y:A, Y:Y or G:A pair at position À1 (this position
should not be occupied by a canonical Watson-
Crick base-pair). The base-pairing partner of pos-
ition À1 does not necessarily belong to the same
loop as the U-turn and can be a distant nucleotide
(e.g. in the 16 S rRNA 1211 motif, the ¯anking
base-pair is 1047:1210, see Figure 1). Tertiary
contacts at positions À1, ‡1, ‡2, and ‡3 will be
investigated.
GNRA patterns
Conserved GNRA patterns occur at 25 positions
in the 16 S and 23 S rRNAs. We de®ne here ``con-
served'' as present in a minimum of 80 % of the
bacterial sequences. GNRA patterns are found
under the following forms.
Canonical GNRA hairpin loops
The canonical form of the GNRA U-turn is the
four-nucleotide hairpin loop, with 13 occurrences
overall (Figure 4 and Table 1, category A). Some of
these candidates have been con®rmed experimen-
tally, including the 23 S rRNA 2659:2662 tetraloop
at the tip of the sarcin-ricin loop (Szewczak &
Moore, 1995; Correll et al., 1998). Comparative
analysis of this loop suggests a base-triple
interaction between position ‡2 (2661) and
the base-pair 2550:2558 (see the CRW site,
http://www.rna.icmb.utexas.edu). Whereas this
base-triple covariation is not consistent with the
loop-loop interaction observed in the recent 50 S
subunit crystal structure (Ban et al., 1999),
rearrangements remain possible and should be
considered in future studies of rRNA dynamics.
The recent low-resolution crystal structure of the
70 S ribosome also suggests an interaction between
the canonical GNRA loop at position 16 S:898-901
and 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 and
23 S:630-633), since both sequences can fold in the
same way (Murphy & Cech, 1994). This is con-
®rmed for the 16 S rRNA loop 727-730 in the
S15,S6,S18 rRNA crystal structure (Agalarov et al.,
2000).
GNRA in larger hairpin loops
Four conserved GNRA motifs occur within hair-
pin loops that contain more than four nucleotides
(Table 1 and Figure 4, category B). While one of
these (23 S:306) is located within a 6 nt loop and is
¯anked on both sides with nucleotides that can
form a non-canonical base-pair, the GNRA
sequence is placed asymmetrically in the other
hairpin loops. In these cases, the loop needs to be
distorted to accommodate a GNRA structure.
Although this is theoretically possible, there is no
experimental precedent. The example at positions
780-784 in 23 S rRNA would have a bulged
K (G or U) following the GNRA structure, while
the two remaining GNRA motifs occur in loops of
size eight, at the second or fourth loop position.
GNRA in internal and multi-stem loops
Eight conserved GNRA sequences occur in
internal or multi-stem loops (Figure 4 and Table 1,
category C). GNRA tetraloop conformations have
never been observed experimentally in such situ-
ations; therefore, these should be considered tenta-
tive. The GNRA sequence is involved in a putative
tertiary interaction in at least one of these loops (see
the CRW site, http://www.rna.icmb.utexas.edu).
The strongest example occurs in the 1108 loop of
16 S rRNA, where position 1109 (‡1) covaries with
the 933:1384 base-pair, while in the 215 loop of 23 S
rRNA, base triple covariations occur at positions ‡1
and À1. Two other internal loops display signi®cant
levels of GNRA/GNRG variation: 16 S rRNA
765-768 (3.4 % GAAG) and 23 S rRNA 818-821
(33 % GAAG) (see above).
UNR patterns
UNR patterns conserved in more than 90 % of
the bacterial sequences are found at 44 sites in 16 S
and 23 S rRNA. We eliminate 11 of these sites that
are ¯anked by Watson-Crick pairs with multiple
compensatory base changes, since we do not
expect U-turns to be enclosed by standard base-
pairs. The remaining 33 sites are ¯anked by
unpaired nucleotides or by a highly conserved
Watson-Crick base juxtaposition (e.g. 95 % U:A)
that could possibly form a non-canonical base-pair.
While the majority of candidates occur in hairpin
or multi-stem loops, where UNR-type U-turns
have already been observed experimentally, three
occur in internal loops (Table 1), an unexpected
and structurally less likely situation. Candidates of
the UNR type were classi®ed into categories D
through J (Table 1).
UNR in trinucleotides hairpin loops
Two trinucleotide hairpin loops contain the UNR
motif directly closed by a single base-pair which is
highly conserved (Figure 4 and Table 1, category
D). The 23 S rRNA base-pair 1082:1086 is U:A in
nearly 100 % of the bacterial and chloroplast
sequences and C:G in almost all of the eukaryotic
sequences (Table 2). Such an atypical base-pairing
constraint 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 the
UAA hairpin were identi®ed in the crystal struc-
ture of the L11 binding region of 23 S rRNA (Conn
et al., 1999; Wimberly et al., 1999). The 1926 triloop
also 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:A
796 U-turns in rRNA

7. and C:G base-pairs do not form identical isosteric
conformations (Gautheret & Gutell, 1997), a pre-
cedent for this type of exchange is the tRNA 15:48
reverse Watson-Crick base-pair. Given similar
sequence constraints in both loops, we expect their
3D structure to be similar as well. Since the bases
‡1 and ‡2 in the 23 S rRNA 1083-1085 loop are
involved in tertiary contacts with base-pairs
1054:1105 and 1055:1104 in the crystal structure of
the L11 binding region of 23 S rRNA (Conn et al.,
1999), we anticipated tertiary interactions at pos-
itions 1927 or 1928. Interestingly, our comparative
analysis revealed a covariation between positions
1928 and 1834 (see Figure 4 and the CRW site,
http://www.rna.icmb.utexas.edu/).
UNR at position 2 flanked by Y:R or
Y:Y base-pairs
Six UNR sites have the UNR pattern at position
2 of a hairpin or internal loop, and ¯anked by a
Y:R or Y:Y juxtaposition (Figure 4 and Table 1, cat-
egory E). This arrangement is similar to the tRNA
anticodon U-turn, except for the difference in loop
sizes. Tertiary contacts at position ‡1 to ‡3 have
been 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-turn
position), 15:1397 (‡1) and 16:920 (‡2). Although
the U-turn position has not been implicated in
long-range tertiary interactions, contacts with
position À1 are possible, as shown in the hammer-
head ribozyme (Pley et al., 1994a) and TcC-loop of
tRNA (Quigley & Rich, 1976). In addition, two of
the U-turn candidates in this category have tertiary
interactions at positions 5 and 6 (23 S rRNA
position 567 and 1065, see Table 1 and Figure 4).
UNR in hairpin loops flanked by G:A base-pairs
Four UNR motifs are ¯anked by a G:A base jux-
taposition (Figure 4 and Table 1, category F). Hexa-
nucleotide loops with the GUAANA sequence
consensus fall in this category, forming a well
characterized three-dimensional motif (Fountain
et al., 1996; Huang et al., 1996; Stallings & Moore,
1997; Puglisi & Puglisi, 1998), with a sheared G:A
closing base-pair and a U-turn forming at the con-
served uridine base. Nucleotides AAU located 3H
to
the 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 is
involved in an important tertiary interaction brid-
ging the 30 S and 50 S ribosomal subunits (Culver
et al., 1999). The 16 S rRNA hairpin loop 691-696
begins with a G and ends with an A and is similar
to the previous two motifs in size and loop closure.
Interestingly, this loop is protected by tRNA
(Moazed & Noller, 1989b) and the association of
subunits (Powers et al., 1993; Merryman et al.,
1999). The fourth motif, 16 S rRNA 260-266, has a
seven-nucleotide loop with a weak covariation in
the Bacteria between positions 260 and 265. This
would create a ®ve-nucleotide hairpin loop with a
G:G, G:A or A:A closing base-pair. These pairing
types can adopt a sheared base-pair conformation
similar to the G:A pair in the other motif.
Sequence variations in the 23 S rRNA 713 loop
are particularly interesting (Table 3). Archaea and
eukaryotes have a central GAAA sequence closed
by a Watson-Crick base-pair (G:C or C:G), while
Bacteria and chloroplasts have a central UNAN
sequence closed by a G:A juxtaposition. Both com-
binations (Watson-Crick pair ‡ GNRA sequence or
G:A pair ‡ UNRN sequence) can form a U-turn at
position 2 of the loop, and thus retain the ability to
form the tertiary interaction with the 30 S riboso-
mal subunit.
UNR in internal and multi-stem loops flanked by
C:G pairs
Three internal and multi-stem loops display con-
served UNA sequences adjacent to a conserved
C: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:G
pairs form in these cases, since the pairs are
¯anked by other secondary structure base-pairs.
Although other non-canonical conformations are
still possible, these three sites are weak U-turn can-
didates. An additional site in this category, found
at 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 positions
1082:1086 and 1925:1929 (only frequencies over 1 % are
shown)
Kingdom Most frequent sequence
A. 1082:1086
(eu)Bacteria U:A (98.8 %) C:G (1.0 %)
(1 phylogenetic event)a
Chloroplast U:A (98.0 %) U:C (2.0 %)
Archaea C:G (59.5 %) U:A (40.5 %)
(3 phylogenetic events)a
Eucarya 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 %)
a
Concerted base changes occurring between closely related
organisms (see Materials and Methods).
Table 3. Sequence variations at 23 S rRNA positions
713-718
Kingdom 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. UNR in loops flanked by other base-pairs
Five UNR sequences are ¯anked by other base
juxtapositions (Figure 4 and Table 1, category H).
Two of these are ¯anked by a secondary structure
base-pair (23 S:1951 and 16 S:1065), but atypical
sequence constraints in these pairs are compatible
with a non-canonical pairing (Table 4). In addition,
the 23 S rRNA 1951 U-turn candidate is associated
to a base-triple type covariation between positions
(1950:1956) and 1954 (Figure 4 and the CRW
site, http://www.rna.icmb.utexas.edu/). The 16 S
Figure 4 (legend shown on page 800)
798 U-turns in rRNA

9. rRNA 787-795 loop contains two overlapping U-
turn signatures: the 95 % consensus for 788-790 is
UYA, while the 83 % consensus for 789-791 is
UAG. The structure of this nine-nucleotide hairpin
resembles the tRNA TcC-loop (Gu et al., 1994),
with a closing A:C base-pair reducing the loop size
to seven. The U-turn occurring at position U55 in
tRNA would be homologous to position U789 in
16 S rRNA (Gu et al., 1994). In addition, in vitro
selection experiments indicate that U789, rather
than U788 is required for ribosome function (Lee
et al., 1997). Therefore, while sequence conservation
alone would favor a U-turn at 788, this turn is
more likely at position 789. Nucleotides within this
Figure 4 (legend shown on page 800)
U-turns in rRNA 799

10. Figure 4. Potential U-turns shown on the E. coli secondary structures for the small subunit (a), the large subunit 5H
half (b) and the large subunit 3H
half (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 interactions
have 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-stem
loops 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 hairpin
loops; E, UNR at position 2 ¯anked by Y:R or Y:Y base-pairs;, UNR in internal and multi-stem loops ¯anked
by 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. loop are protected by ribosomal subunit associ-
ation, suggesting that this U-turn motif is involved
in tertiary interactions (Powers et al., 1993;
Merryman et al., 1999).
UNR in loops without flanking base-pair
Flanking base-pairs are unknown or ambiguous
for ten of the U-turn candidates (Figure 4 and
Table 1, category I). Four of these potential U-turns
are associated with predicted tertiary interactions
at 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 at
positions 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 (Doring
et al., 1991). These tertiary interactions correspond
to positions ‡2 and ‡3 after the proposed U-turn.
Tertiary base covariations are also observed at the
3H
end of the 23 S rRNA 1339 loop (pseudoknot
1343-1344:1403-1404, see the CRW cite) and in the
23 S rRNA 2583 loop (1782:2586). Both of these
proposed interactions are supported experimen-
tally, the former by site-directed mutagenesis (Kooi
et al., 1993), and the latter by U.V. crosslinks
between positions 2584-2588 and 1777-1792 (Stiege
et al., 1983). The 23 S:2497 loop has putative
tertiary interactions 5H
to the turn at positions À1
and À2 (2499:2453 and 2498:2454).
Ambiguous UNR/GNRA exchanges
Exchanges between UNR and GNRA sequences
occur at two hairpin loop sites (16 S:863 and
16 S:1013, see Figure 4). This is similar to the
sequence variation at the 23 S:713 hairpin loop
(Table 3). This type of variation could result from a
selective pressure for U-turns at these sites. The
16 S:863 loop has another characteristic associated
with U-turns: two positions 3H
to this putative U-
turn form tertiary pseudoknot base-pairs to 16 S
rRNA positions 570-571 (Gutell et al., 1986; Vila
et al., 1994). However, in both cases the UNR
sequence 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 are
uncertain, since these pairing types have not been
observed ¯anking a U-turn.
Conclusion
Comparative sequence analysis enables us to dis-
tinguish randomly occurring U-turn signatures
from candidates that are supported by sequence
conservation and speci®c patterns of base-pair
exchanges. We have identi®ed 58 UNR and GNRA
U-turn candidates in a variety of structural settings
in the 16 S and 23 S rRNAs. Since the sequence
and structural information that de®nes a U-turn is
minimal and the sequence constraint rules that we
have used to identify U-turns may be associated
with other structural motifs, some of our predicted
U-turns may be incorrect. Alternatively, these U-
turn signatures may be associated with structural
conformations that alternate between U-turns and
these other structural motifs. Used as working
hypotheses, putative U-turns and the associated
tertiary interactions can be used for modeling
(prior to re®nement) and interpretation (after
re®nement) of the X-ray crystal structures of the
ribosome.
Materials and Methods
We have used the alignments of small and large sub-
unit rRNA sequences maintained by us at the University
of Texas (R.R.G., unpublished results). The small subunit
rRNA alignment contains 5826 Bacteria, 182 chloroplast,
264 Archaea and 1054 Eukaryotic sequences. The large
subunit rRNA alignment contains 326 Bacteria, 103
chloroplast, 41 Archaea and 263 Eukaryotic sequences.
Secondary structure diagrams for representatives of the
main phylogenetic groupings are inferred with compara-
tive sequence analysis (Gutell et al., 1993; Gutell, 1994)
and are available from our Austin, Texas CRW site (The
Comparative RNA Web Site: http://www.rna.icmb.u-
texas.edu/, R.R.G., unpublished results).
Base frequencies were computed independently in the
Bacteria, chloroplast, Archaea and Eukaryotic align-
ments. When not otherwise speci®ed, base or base-pair
frequencies refer only to Bacteria sequences. Base num-
bering always refers to Escherichia coli 16 S or 23 S rRNA
sequences (GeneBank accession no. J01695). The phyloge-
netic events for base-pairs in Tables 2 and 4 were
derived from the CRW site (http://www.rna.icmb.
utexas.edu/). Here, the numbers of mutual changes that
have occurred throughout evolution for each pair in our
comparative structure model are accessible, as well as
Table 4. Sequence variations at base-paired positions
16 S rRNA 1064:1192 and 23 S rRNA 1950:1956 (all
frequencies over 1 % are shown, see the CRW web site
for a detailed analysis)
Kingdom Most frequent sequence
A. 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)a
B. 23 S 1950:1956
(eu)Bacteria G:U (94 %) U:A (5 %)
(2 phylogenetic events)a
Archaea G:U (59 %) U:G (17 %) A:A
(15 %) U:A (10 %) (no
phylogenetic event)a
Eucarya C:A (89 %) G:U (3 %) U:G (3 %)
U:U (3 %) (2 phylogenetic
events)a
a
Concerted base changes occurring between closely related
organisms (see Materials and Methods).
U-turns in rRNA 801

12. details of the base-pair types and speci®c phylogenetic
location for each mutual change. A ``phylogenetic event''
was recorded when both positions in the pair varied
between two consecutive organisms. This approximation
is simplistic but conservative, since all but the most
recent events are neglected.
tRNA base frequencies, were derived from the 1997
version of M. Sprinzl's tRNA alignments (Sprinzl et al.,
1991). All nuclear tRNAs and tDNAs were included in
our base counts. The Yeast tRNAPhe
numbering is used
throughout.
The Figures and Tables for this article are available
online at the main CRW site (http://www.rna.icmb.u-
texas.edu/, go to ``RNA Structure Analysis/U-Turn'') or
by using the speci®c URL (http://www.rna.icmb.utexa-
s.edu/ANALYSIS/U-TURN/).
Acknowledgments
This work was supported in part from the NIH grants
awarded to R.G. (NIH - GM48207) and startup funds
from the Institute for Cellular and Molecular Biology at
the University of Texas at Austin.
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Edited by J. A. Doudna
(Received 25 February 2000; received in revised form 22 May 2000; accepted 22 May 2000)
U-turns in rRNA 803