Article No. mb982106
J. Mol. Biol. (1998) 283, 571±583
A Common Motif Organizes the Structure of
Multi-helix Loops in 16 S and 23 S Ribosomal RNAs
Neocles B. Leontis1 and Eric Westhof2*
1
Chemistry Department
Bowling Green State
University, Bowling Green
OH 43403, USA
2
Institut de Biologie
MoleÂculaire et Cellulaire du
CNRS, ModeÂlisations
et Simulations des Acides
NucleÂiques, UPR 9002
15 rue Rene Descartes
F-67084 Strasbourg Cedex
France
Phylogenetic and chemical probing data indicate that a modular RNA
motif, common to loop E of eucaryotic 5 S ribosomal RNA (rRNA) and
the a-sarcin/ricin loop of 23 S rRNA, organizes the structure of multihelix loops in 16 S and 23 S ribosomal RNAs. The motif occurs in the 30
domain of 16 S rRNA at positions 1345-1350/1372-1376 (Escherichia coli
numbering), within the three-way junction loop, which binds ribosomal
protein S7, and which contains nucleotides that help to form the binding
site for P-site tRNA in the ribosome. The motif also helps to structure a
three-way junction within domain I of 23 S, which contains many universally conserved bases and which lies close in the primary and secondary
structure to the binding site of r-protein L24. Several other highly conserved hairpin, internal, and multi-helix loops in 16 S and 23 S rRNA
contain the motif, including the core junction loop of 23 S and helix 27 in
the core of 16 S rRNA. Sequence conservation and range of variation in
bacteria, archaea, and eucaryotes as well as chemical probing and crosslinking data, provide support for the recurrent and autonomous existence
of the motif in ribosomal RNAs. Besides its presence in the hairpin ribozyme, the loop E motif is also apparent in helix P10 of bacterial RNase P,
in domain P7 of one sub-group of group I introns, and in domain 3 of
one subgroup of group II introns.
# 1998 Academic Press
*Corresponding author
Keywords: RNA; multi-helix loops; recurrent motif; phylogenetic analysis
Introduction
Single-stranded RNA molecules fold on themselves to form short double helices consisting of
classical Watson ±Crick base-pairs frequently interrupted by stretches of ``mismatched'' bases, comprising so-called internal loops, hairpin loops, and
multi-helix junction loops (Fresco et al., 1960). Universally conserved, nominally unpaired nucleotides often occur within these loops. That many of
these regions are in fact highly structured was ®rst
clearly indicated by chemical probing experiments
of 16 S rRNA (Moazed et al., 1986).
Phylogenetically based covariation analysis is
the most reliable means for determining secondary
structure in homologous RNA molecules (Michel
Abbreviations used: rRNA, ribosomal RNA; r-protein,
ribosomal protein; bp, base-pair; PSTV, potato spindle
tuber viroid; R, purine; Y, pyrimidine; CMCT,
1-cyclohexyl-3-(2N-morpholinethyl)-carbodiimide-methop-toluene sulfonate.
E-mail addresses of the corresponding authors:
[email protected] and
[email protected]
0022±2836/98/430571±13 $30.00/0
et al., 1982; Woese & Pace, 1993). It can also be
used to identify nucleotides involved in tertiary
interactions, when these co-vary in a Watson±
Crick pairing fashion, but not when these involve
universally conserved, single-stranded regions of
the already deduced secondary structure (Woese &
Gutell, 1989; Larsen, 1992). The modular approach
to sequence analysis of RNA is based on the
assumption that recurrent, conserved elements of
primary and secondary structure share common
three-dimensional (3D) structures and therefore
constitute building blocks for organizing the tertiary structure of RNA (Michel & Westhof,
1990). Such elements must be viewed as structural
units or motifs, since they comprise highly conserved nucleotides, which are paired non-canonically and which therefore do not co-vary in a
Watson± Crick fashion. Identi®cation of such
motifs within internal loops, followed by structural
characterisation using NMR and X-ray crystallography, advances our ability to model the secondary and tertiary architecture of native RNA
molecules, by elaborating a ``structural vocabulary'' applicable in evaluating possible interactions
in any structured RNA.
# 1998 Academic Press
572
Potential structural motifs are easiest to identify
when the conserved sets of nucleotides occur as
internal loops, bracketed on either side by
Watson ±Crick helices or hairpin loops. Such is the
case of the loop E motif ®rst identi®ed in 1985 as a
distinctive RNA structural motif when it was
found that very similar, conserved, asymmetric
internal loops in eucaryotic 5 S rRNA and potato
spindle tuber viroid (PSTV) shared an exceptional
propensity for UV crosslinking between speci®c
guanosine and uridine residues, as shown in
Figure 1A (Branch et al., 1985). The motif was also
Recurrent Motif in Multi-helix Loops in RNA
found in a conserved hairpin loop in domain VI of
23 S-like rRNA molecules (residues 2654± 2666 of
Escherichia coli 23 S and 4218 ± 4330 of rat 28 S
rRNA), which is the site of speci®c RNA modi®cation by the cytotoxic proteins a-sarcin and ricin
(Endo et al., 1993) and is directly involved in binding of elongation factors by the ribosome (Moazed
et al., 1988). It has been pointed out that the
element is likely to be widespread (Shen et al.,
1995) and that it may exist in other hairpin/stem
loops in 23 S rRNA (Wimberly et al., 1993;
Wimberly, 1994). UV crosslinking showed that the
Figure 1. A, Molecules in which the ``loop E motif'' has been established by NMR or UV cross-linking. The conserved nucleotides of the motif are boxed. Asterisks designate the G and U residues cross-linked upon irradiation
with UV light. From left to right: loop E from eucaryotic 5 S rRNA (305 sequences superimposed on that of X. laevis).
Loop E from Euryarchaeal 5 S rRNA (36 sequences superimposed on that of Methanococcus vanniellii). The 5 S
sequences are numbered according to the 5 S of X. laevis. sarcin/ricin stem from 23 S rRNA (sequence variations
from all three phylogenetic groups superimposed on E. coli sequence and numbering). Conserved core from PSTV
(Branch et al., 1985). Loop B from the hairpin ribozyme (Butcher & Burke, 1994a). Red letters: conserved nucleotides.
Green: base-pairs showing Watson ± Crick variation. Blue: residues within motif showing variation (for sarcin/ricin
stem see Table 1; for euryarchaeal 5 S rRNA: 13 A A, 4 G A, 5A G, 15 U A). Black: Other variable positions. B, Loop
E motifs in internal or hairpin loops of 23 S rRNA and 16 S rRNA. From left to right: 23 S rRNA helix 11; 23 S rRNA
helix 13; 23 S rRNA internal loop between helices 31a and 32. 16 S rRNA helix 27 in core region joining the 50 and
the central domains of the molecule. Residues are colour-coded as in A. Variations observed in three main phylogenetic groups superimposed on E. coli sequence and numbering system.
Recurrent Motif in Multi-helix Loops in RNA
motif exists in loop B of the hairpin ribozyme from
tobacco ringspot virus (Butcher & Burke, 1994a).
Crosslinked molecules retain enzymatic activity
(Butcher & Burke, 1994a; Burke et al., 1996). Here
we present evidence indicating that the motif is
not limited to hairpin stems or internal loops, but
also occurs in multi-helix junctions at critical sites
in the large ribosomal RNAs.
Results
Structural description of the motif
Both the sarcin/ricin loop from 23 S and the Eloop from 5 S rRNA have been studied as oligonucleotide fragments by high-resolution NMR, from
which very similar three-dimensional structures
were derived for the conserved nucleotides of the
motif (Wimberly et al., 1993; Szewczak & Moore,
1995). The structures also agree with the extensive
chemical probing data available for X. laevis 5 S
rRNA (Romaniuk et al., 1988; Westhof et al., 1989;
Romby et al., 1990). The salient structural features
identi®ed by NMR are shown in Figure 2. They
573
are: (1) a sheared A G pair; (2) a trans-Hoogsteen
U A pair; (3) a bulged G positioned in the deep
groove; and (4) a trans (locally parallel)-HoogsteenHoogsteen A A pair (boxed residues in Figure 1).
The sheared pairing of the conserved A G pair
(A77 G99 in the Xenopus sequence, A106 G130 in
the universal alignment) involves hydrogen bonding between AN6 and GN3 and between AN7 and
GN2. This leaves AN1 very exposed and reactive
towards DMS, whereas GN1 is partly exposed and
only reactive under semi-denaturing conditions in
X. laevis 5 S rRNA. Neither AN7 nor GN7 is reactive under native conditions. In the conserved,
trans-Hoogsteen U76 A100 (Xenopus numbering,
U104 A132 universal alignment), UN3 is Hbonded to AN7 leaving the Watson± Crick site
(N1) of A100 exposed and reactive while UN3 and
AN7 are protected in this pair. The N1 of the
bulged G75 (G103) is likely to be H-bonded to the
phosphate oxygen of A101 (A132), consistent with
observed lack of reactivity toward CMCT. Its N7,
however is completely exposed, as shown by the
fact that it is the most reactive base in 5 S rRNA
towards a sterically bulky nickel-organic reagent
Figure 2. Stereo view of the 3D structure of the sheared A G, trans-Hoogsteen U A, bulged G, and parallel A A
base-pairs of the loop E motif as determined by high-resolution NMR (Szewczak & Moore, 1995). The hydrogenbonding in these base-pairs is depicted to the right.
574
(Zheng et al., 1998). The direction of the ribose
phosphate chain is locally reversed at A74 (A102)
resulting in a symmetrical pairing between A74
and A101 (A133), which involves H-bonding of N7
and N6 of one adenine with N6 and N7 of the
other. Both bases are found to be reactive at N1
and unreactive at N7 in Xenopus 5 S rRNA.
On the basis of these structural features, one
expects to observe the following sequence variation
where the motif is evolutionarily conserved
(Leontis & Westhof, 1998): the sheared A G is
expected to covary with a sheared A A (Cate et al.,
1996a) but neither with the non-isosteric opposite
sheared G A pair nor with the G G opposition,
which would require another pairing geometry.
The U A pairing should be strictly conserved.
On the other hand, signi®cant variation is
expected at the position of the locally parallel
A A pair. One expects to see A G as well as
G A Hoogsteen-Hoogsteen (but no G G oppositions again). All R Y, Y R, and Y Y combinations can be accommodated by adopting a
trans-Watson± Crick geometry, while maintaining
the locally parallel strand orientation.
Recurrent Motif in Multi-helix Loops in RNA
Table 1. Sequence variation at the parallel base-pairs
(positions 2654 and 2666 in the E. coli sequence) in the
loop E motif of sarcin/ricin loop of 23 S rRNA in three
phylogenetic groups and mitochondria
Note the predominant pairing is AC in bacteria and mitochondria but AA in eucaryotes and Archaea and that most
variants are related to the dominant pair within a given phylogenetic group by a single mutation. (Number of sequences: 180,
bacterial; 64, eucaryotes; 24, archaeal; and 144, mitochondrial.)
Sequence variation in established
loop E motifs
To establish criteria for evaluating candidate
loop E motifs more precisely than by consensus
sequence comparisons, we examined the sequence
variations in the best-characterized molecules
known to harbour the motif, the sarcin/ricin stem
of 23 S rRNA and loop E of 5 S rRNA (Figure 1A).
We found that the motif exists not only in eucaryotic 5 S rRNA but also in the 5 S of the Euryarchaeota subgroup of Archaea. The sarcin/ricin stem
exists in the 23 S-like RNA of all three phylogenetic groups and of mitochondria. The bulged G,
trans-Hoogsteen U A and sheared A G pairs are
100% conserved in both 5 S groups. The only
exceptions in the sarcin/ricin stem are two mitochondial sequences, which have A A in place of
A G. As discussed above, A A can also form a
sheared base-pair isosteric to a sheared A G bp.
The A G and U A pairings are also conserved in
the viroid and hairpin-ribozyme versions of the
motif. The bulged base is a C in the viroids (Keese
& Symons, 1985) and a U in the hairpin ribozyme.
The symmetrical A A pairing is conserved in all
eucaryotic 5 S rRNAs. In 5 S of Euryarchaeota,
however, A A, A G, GA and U A are also
observed for this pairing. A A is the most common
opposition at the equivalent positions of eucaryotic
and archaeal sarcin/ricin stems, while A C is the
most common in bacterial and mitochondrial sarcin/ricin stems. The variations at this position in
sarcin/ricin loops are summarized in Table 1. The
variations observed in 5 S loop E and sarcin/ricin
loops fall into three groups: (1) R R (A A, A G,
G A, but never G G), (2) R Y and Y R (A Y and
Y A, but rarely GY or Y G), and (3) Y Y (U U,
U C). All observed R R pairings are compatible
with trans-Hoogsteen-Hoogsteen, as seen in the
NMR structure for A A. The other two groups are
both compatible with trans Watson± Crick/
Watson± Crick pairing. Next to the symmetrical
pair, another non-canonical pairing is found in
both 5 S loop E and the sarcin/ricin stem. In some
phylogenetic groups only Y Y pairings are found
here, while in others, wobble pairs (including A C
and C A) and Watson ± Crick pairs also occur. The
next base-pair is usually Watson± Crick, but in
some groups Y Y and wobble pairs are among the
variations. A Watson± Crick base-pair, which in
the sarcin/ricin site is a conserved C-G in all
phylogenetic groups, always follows the sheared
A G pair. Thus, Watson± Crick pairs ¯ank both
sides of the motif.
To summarize, the observed sequence variation
agrees with stereochemical expectations based on
the 3D structures. The U A is universally conserved, owing to the unique trans-Hoogsteen
pairing. The bulged base is not restricted to G.
Any pairing compatible with a trans-parallel
orientation of the bases is allowed immediately 50
to the bulged base. No insertions are observed
within the region bracketed by the parallel pair
and the sheared pair. In addition, it is observed
that the ®rst Watson± Crick pairing occurs at a
variable distance from the parallel base-pair; base
insertions or deletions may also occur here. The
data are insuf®cient yet for understanding the
possible structural constraints underlying these
sequence variations, although they do point to a
role akin to a ¯exible joint (see below). In the
575
Recurrent Motif in Multi-helix Loops in RNA
hairpin ribozyme, the ®rst Watson± Crick pairing
is several non-canonical pairings removed from
the loop E motif.
Loop E motifs in internal and hairpin loops of
23 S rRNA
Three hairpin or internal loops in addition to the
sarcin/ricin hairpin stem were identi®ed in 23 S
rRNA as loop E sites which meet these criteria.
These occur in the highly conserved helices 11 and
13 in domain I and in the internal loop between
helices 31a and 32 in domain II, in the 50 -most part
of the 23 S molecule. (The positions of the proposed loop E sites discussed in this paper within
the conventionally drawn secondary structures of
16 S and 23 S rRNA are shown in Figure 3). The
possible existence of loop E motifs in helices 11, 13,
and in the core of 23 S (see below) was previously
suggested, based on consensus sequence comparisons (Wimberly et al., 1993; Wimberly, 1994). The
E. coli versions of these are shown in Figure 1B, on
which sequence conservation and variations
observed in all three phylgenetic groups are superimposed. Very few deviations from the criteria
de®ned above are observed and these are restricted
to speci®c phylogenetic groups. These examples
show that the motif may exist in either orientation
closing a hairpin loop, which gives additional support for its autonomous existence. Footprinting
data are also consistent with existence of the motif
at these positions, as summarized in Table 2
(Egebjerg et al., 1987, 1990).
A loop-E motif in the core of 16 S rRNA
Universally conserved nucleotides 889-892/907909 comprise a loop E motif in a hairpin stem
(helix 27) in the core of 16 S rRNA (rightmost
panel, Figure 1B). The sheared pair is A A, with a
small number of A G in eucaryotes. The U A is
invariant in all groups. The bulged G shows some
variation (A, C, or U) and the symmetrical A A, a
very small number of AG, GA and AU variants.
Watson ±Crick or wobble pairs separate the motif
from a conserved GCAA hairpin loop; the pair
adjacent to the sheared A R pair is a conserved
Y-R pairing (C-G in all Archaea, U G in eucaryotes, C-G, U G or C A, in bacteria). Pairing of
positions 912± 910 with 885 ± 887 was recently proposed on the basis of site-directed mutagenesis,
completing helix 27 (Lodmell et al., 1995). This
leaves base 888 unpaired, but does not hinder formation of the motif. Helix 27 is part of a complex
junction, which includes two more helical elements
and a pseudo-knot to organize the 50 and central
domains of 16 S rRNA (Masquida et al., 1997). The
nucleotides of the loop E motif in helix 27 are protected from chemical probes both in the naked 16 S
rRNA and in the 30 S subunits (Moazed et al.,
1986). During the ®rst stages of in vitro assembly of
the RNA with r-proteins, residues G886 G890, on
the 50 side of the Loop E motif become more reac-
tive to chemical probes, indicating that a proteinmediated conformational change probably occurs
(Stern et al., 1988b). It was recently proposed that a
conformational switch occurs during translation in
which nucleotides 912 ±910 alternately pair with
885-887, as in Figure 1B, or with 888-890 (Lodmell
& Dahlberg, 1997). In the latter conformation the
loop E motif would be disrupted by pairing of the
bulged G890 with C910 and of the reversed A889
with U911. However, in isolated wild-type ribosomes, the predominant conformation is that
allowing formation of the loop E motif (Lodmell &
Dahlberg, 1997). Further, mutations favouring that
conformation are always less severe than those stabilizing the alternative conformation. It can be
added that helix H27 can be drawn with a loop E
motif in 16 S rRNA from mitochondria as well.
Interestingly, a conformational switch between a
structure involving a GNRA tetraloop and one
with a loop E motif occurs during the pathway of
the PSTV viroid processing (Baumstark et al.,
1997).
Three-way junctions organized by loop
E motifs
Three-way junctions occur widely in stable RNA
including ribosomal RNAs (5 S, 16 S and 23 S),
ribozymes, and viral RNAs. RNA three-3-way
junctions typically have a number of unpaired or
non-canonically paired bases in one or more
strands linking and organizing the helices in space,
as exempli®ed in the structure of the hammerhead
ribozyme (Pley et al., 1994). One of several threeway junctions which are found in the 30 major
domain of 16 S RNA binds S7 (Chiaruttini et al.,
1989; Dragon & Brakier Gingras, 1993) and forms
part of the P-site for tRNA binding (Moazed &
Noller, 1986). This junction is usually drawn in
secondary structure maps with 19 unpaired
nucleotides, many of which are universally conserved or show very restricted variation (Figure 3).
In fact, nucleotides 1345 ±1349 and 1373± 1376 form
a loop E motif bridging helices 29 and 43, which
together with helix 28 comprise the junction
(Figure 4A). The only variable position within the
motif is the parallel pairing between positions 1346
and 1375. This is a highly conserved A A in bacteria and in Archae (no exceptions), while mainly
A U in eucaryotes with variants A C, A A, C U,
G U, U A, C A, and U C, consistent with maintenance of the trans-trans pairing at this position.
A conserved U U (Y U in eucaryotes) can form
between positions 1345 and 1376. Usually U1345 is
shown Watson± Crick paired with conserved A938
in secondary structures of 16 S. Watson± Crick bp
1350 ± 1372 and 1344 ±939 ¯ank the motif. The exact
position of the three-way junction branch point
depends on how U1345 is paired, but in either case
the branching does not disrupt the motif. Chemical
probing of the Watson± Crick sites of nucleotides
in naked 16S rRNA showed that A1346, A1349,
A1374, A1375 within the motif are reactive,
Figure 3. Conventional secondary structures of 16 S rRNA and domains I and II of 23 S rRNA showing locations of proposed loop E sites. 16 S rRNA sites: helix 27 in
the core region (see also Figure 1B) and the three-way junction comprising helices 28, 29, and 43 in the 30 -major domain (see also Figure 4A). 23 S rRNA sites: internal loops
at helices 11, 13, 31a and 32 (see also Figure 1B); three-way junction formed by helices 3, 4, and 23 (see also Figure 4B); multi-helix loops adjacent to helices 21 and 36 (see
also Figure 5B). Not shown in this ®gure: the loop E motifs located in the sarcin/ricin stem in domain VI (see Figure 1A) and in the core region of 23 S rRNA (see
Figure 5A).
577
Recurrent Motif in Multi-helix Loops in RNA
Table 2. Chemical probing data of proven and proposed loop E motifs
Base-pair
Base-atom
5 S rRNA
23 S S/R helix
23 S helix 11
23 S helix 13
23 S loop 31a/32
16 S 3WJ 28/29/43
23 S 3WJ 3/4/23
23 S core
23 S MWJ 21
23 S MWJ 36
A-N1
R
r
R
r
r
R
r
r
R
r
Sheared
A-N7 G-N1
U
U
U
U
r
U
U
U
U
r
U
r
G-N7
U
R
UA trans-Hoogsteen
U-N3 A-N1 A-N7
U
U
U
U
U
r
r
r
U
r
R
r
r
r
r
R
r
r
U
r
U
U
Bulged G
G-N1 G-N7
U
U
U
r
ra
U
r
R
r
U
R
r
Parallel
A1-N1 A1-N7 A2-N1 A2-N7
R
r
r
r
Ub
R
r
U
U
Ub
U
U
R
r
r
r
r
R
R
r
U
r
U
U
R, very reactive; r, weakly reactive; U, unreactive. Blanks indicate undetermined. 3WJ stands for three-way junction and MWJ for
multi-way junction. The numbers indicate helices adjacent to the motifs identi®ed (see also Figures 3 to 5).
a
The bulged base is A in the probed sequence.
b
The pair is G A in the probed sequence. Probing data for 5 S rRNA are from Westhof et al. (1989), for 16 S from Moazed et al.
(1986) and Baudin et al. (1987), and for 23 S from Egebjerg et al. (1990).
whereas bulged G1347, U1348 and G1373 are not
(Moazed et al., 1986), in complete agreement with
the probing data of the corresponding positions of
5 S loop E discussed above (see Table 2). In the
30 S particle, these bases become protected from
reactive probes, due to the binding of protein S7
(see below).
A three-way junction in domain I of 23 S RNA
The three-way junction formed by helices 3, 4,
and 23 in domain I of 23 S rRNA also contains a
loop E motif. As in the 16 S junction, the motif
bridges two of the helical arms (helices 3 and 23)
creating potential continuous stacking between
them (Figure 4B). The branch point of the junction
occurs at the equivalent position: two pairings
from the bulged base or one after the A A pair.
However, the two junctions differ in the direction
of branching. In the 23 S three-way junction,
branching occurs in the strand containing the
unpaired G, while it was the opposite in the 16 S
junction, stressing again the autonomous existence
of the motif. Very little sequence variation is
observed across three phylogenetic groups.
Chemical probing of naked 23 S rRNA and 50 S
subunits supports the existence of the junction
(Table 2) (Egebjerg et al., 1990). A stretch of ®ve
other nominally unpaired bases within this
junction loop is also universally conserved
(G446 G450). The loop E motif in this junction
may, as in the 16 S three-way junction, organize
highly conserved single-stranded residues for
speci®c interactions with other RNA sequences.
The primary binding protein L24 binds to 23 S
rRNA at two sites within domain I, one of
which is adjacent in the primary and secondary
structure to this three-way junction (Egebjerg
et al., 1987). Thus the Loop E motif may also
help to structure the neighbouring region for
RNA ± protein interactions.
A loop E motif in the core of 23 S rRNA
The core of 23 S rRNA is closed by a helix
formed by the 50 and 30 termini of the molecule; it
is usually drawn in a circular format with the six
domains radiating outward (Airport Terminal
model, see Figure 5A). Covariation analysis
detected Watson± Crick interactions between positions 1262± 2017, 1269 ± 2011, and 1270 ±2010 in
23 S rRNA (Gutell & Woese, 1990), suggesting that
an irregular helix forms between nucleotides 1262
and 1270, which link domains II and III, and residues 2010 and 2017, which link domains IV and V
(Haselman et al., 1989). In fact these nucleotides
comprise a loop E motif, highly conserved across
all three phylogenetic groups and also found in
the highly reduced mitochondrial sequences
(Figure 5A). The only sequence variations occur at
the symmetrical A A parallel pairing which is conserved in bacterial and archaeal sequences but displays three C A and one A G variants in
eucaryotes. The folding of the motif creates a
stretch of continuous base-pairing between
domains II and IV. Disruption of either Watson±
Crick basepair 1262± 2017 or 1269± 2011, ¯anking
the motif was shown by site-directed mutagenesis
to impair the ability of the 23 S to engage in protein synthesis (Aagaard & Douthwaite, 1994). All
adenosine residues in the motif were found to be
slightly reactive with DMS in the naked 23 S
rRNA. Double-helix speci®c CV nuclease cut
strongly at the Watson± Crick sites ¯anking the
motif and weakly within it, giving further proof
for its existence. The motif is completely protected
from chemical and enzymatic probes in the 50 S
ribosome, consistent with multiple RNA-RNA or
RNA-protein interactions in the folded RNA.
Loop E motifs in multi-helix loops in domain I
and II of 23 S
A loop E motif conserved in all three phylogenetic groups is found at positions 1186± 1190/817±
578
Recurrent Motif in Multi-helix Loops in RNA
Figure 4. A, Three-way in 16 S rRNA comprising helices 28, 29, and 43, which contains a loop E motif in all phylogenetic groups. Left: as usually drawn. Right: redrawn to highlight loop E motif bridging helices 29 and 43 (note
rotation of junction relative to conventional view). Residues coloured as in Figure 1A. Variations observed in all three
phylogenetic groups superimposed on E. coli sequence and numbering system. A A pair in motif is conserved in bacteria and Archaea, but is predominantly A U in eucaryotes with variants A C, A A, C U, GU, U A, C A, and U C.
Box indicates inserted C present in eucaryotic and archaeal sequences. B, Three-way junction in domain I of 23 S
rRNA comprising helices 3, 4, and 23 containing Loop E motif. Left: as usually drawn. Right: redrawn to highlight
loop E motif bridging helices 3 and 23 (note rotation of junction). Residues colored as in Figure 1A. Variations
observed in all three phylogenetic groups superimposed on E. coli sequence and numbering system.
821 in E. coli 23 S at the base of helix 36, which
connects the seven-way junction loop of domain II
to the rest of the molecule. The motif creates potential stacking between helix 36 and either helix 37 or
helix 45. The branch point occurs next to the parallel base-pairs and does not interrupt the motif. The
parallel pair is predominantly A A in eucaryotic
23 S, G A in bacteria, and mixed in Archaea.
Another loop E motif is found in the variable junction in domain I adjacent to helix 21. Sequence
variations for both motifs are superimposed on the
E. coli sequence (Figure 5B) and chemical probing
data are summarized in Table 2 (Egebjerg et al.,
1990).
Loop E motifs in catalytic RNAs
Besides ribosomal RNAs and the other known
examples, PSTV and the hairpin ribozyme, the
loop E motif probably exists in other structured or
catalytic RNAs. The loop E motif has thus been
identi®ed in a subfamily of type B RNase P
sequences, in helix P10.1, where it replaces a regular Watson± Crick helix (Massire et al., 1998). The
loop E motif is also apparent in the P7.1 ± P7.2 subdomain of subgroup IA2 of group I introns (Michel
& Westhof, 1990) and in domain 3 of one class of
group II introns (Michel et al., 1989). These motifs
are illustrated in Figure 6.
579
Recurrent Motif in Multi-helix Loops in RNA
Figure 5. A, Core region of 23 S rRNA containing loop E motif. Left: schematic view as conventionally drawn.
Right: redrawn highlighting the loop E motif, which potentially bridges helix 26 of domain II and helix 61 of domain
IV. Residues colored as in Figure 1A. Variations observed in all three phylogenetic groups superimposed on E. coli
sequence and numbering system. B, Multi-helix junctions from 23 S rRNA containing loop E motifs. Left: loop E
motif at base of helix 21 in multi-helix loop in domain I. Bacterial and archaeal sequences only (eucaryotic sequences
do not retain the AG sheared pairing). Right: loop E motif in seven-way junction in domain II of 23 S rRNA, conserved in three phylogenetic groups. The motif potentially bridges helices 36 and 37 (as shown) or helices 36 and 45.
Unpaired G is conserved in eucaryotes and Archaea, but may be G, A or U in bacteria. The parallel pair is predominantly G A in Bacteria and Archaea and A A in eucaryotes. Other variants: bacteria: A A and G C; eucaryotes: G A
and U A; Archaea A A, C A, G U, U A and one G G. Residues colored as in Figure 1A.
Discussion
Evolutionary implications
Why are there so many loop E sites in the large
RNAs, and why are these so highly conserved?
The fact that they are conserved in all phylogenetic
groups indicates that loop E motifs were present in
the Ur-ribosome. Their occurrence at sites involved
in binding primary proteins and RNA sequences
which de®ne the domain structure of 16 S and 23 S
rRNA suggests that they evolved within internal
and junction loops due to their special ability to
participate in the organization and structuring of
multiple junctions as well as in RNA-RNA and
RNA-protein interactions. The fact that the Watson ±Crick sites of all the adenosine residues in the
motif are available for interaction is noteworthy
and parallels the situation found in the GNRA
loop motifs and their receptors (Jaeger et al., 1994;
Cate et al., 1996b).
Protein binding by loop E motifs
It is well known that loop E of eucaryal 5 S
rRNA forms part of the binding site for ribosomal
protein L5 (Allison et al., 1991) and transcription
factor TFIIIA (Romaniuk, 1989), while elongation
factors and cytotoxins interact with the sarcin/ricin
loop of 23 S rRNA. The bulged G must be present
580
Recurrent Motif in Multi-helix Loops in RNA
Figure 6. Loop E motifs identi®ed in ribozymes other than the
hairpin ribozyme (see Figure 1A).
From left to right: group I ribozyme, subgroup A2, P7.1 and 7.2
extension (Michel & Westhof,
1990); group II, subgroup a,
domain III (Michel et al., 1989); and
RNase P, type B, loop L10.1
(Massire et al., 1998).
for speci®c recognition and modi®cation of the
RNA motif by a-sarcin (Wool et al., 1992). The
interaction of elongation factors EF-G and EF-Tu
with the sarcin/ricin loop of 23 S rRNA was
demonstrated using chemical footprinting (Moazed
et al., 1988). Protection was observed upon binding
of EF-Tu or EF-G within the motif at the bulged
G2655 and at A2665, which is trans-Hoogsteen
paired to U2656. In the absence of factors, the
Watson ±Crick faces of A2665 and G2655, but not
A2654 (which probably interacts with C2666 in the
E. coli sequence) are reactive.
S7 is the sole primary binding protein in the 30
domain of 16 S. Foot-printing and cross-linking
experiments show that nucleotides comprising the
loop E motif of the 16 S three-way junction in
Figure 4A form part of the binding site of this protein (Wower & Brimacombe, 1983; Powers et al.,
1988). Concise footprints obtained using solventgenerated hydroxyl radicals show that S7 protects
nucleotides on the 30 side of the loop E motif of the
three-way junction, and in helices 29 and 43, which
are bridged by the motif (Powers & Noller, 1995).
The 50 -stagger of six to seven nucleotides observed
in the footprint is consistent with axial binding to
the deep groove of the RNA. The remaining
unpaired bases of the three-way junction are
lightly protected from hydroxyl radicals by S7.
However, some nucleotides become more accessible upon protein binding. Most notably, U1381,
adjacent to the loop E motif becomes reactive to
kethoxal upon binding by S7 and is strongly reactive in 30 S subunits. It is protected by tRNA binding to ribosomes (Moazed & Noller, 1986; Moazed
et al., 1986; Powers et al., 1988). These data suggest
that the loop E motif of this three-way junction
may play two roles: to provide a binding site
for S7, which helps to ®ne-tune the local RNA
structure, and to interact with other nucleotides in
the junction or in the nearby four-way junction to
help form the binding site for the tRNA anti-codon
loop. The structure of S7, recently solved using
X-ray crystallography (Hosaka et al., 1997;
Wimberly et al., 1997), indicates extensive protein
surface for interaction with double-helical RNA.
The majority of loop E motifs found in 23 S
rRNA occur in the 50 half of the molecule. In fact,
four occur in domain I, the ®rst to be transcribed
and the ®rst to fold (Tumminia et al., 1994). Chemical and enzymatic probing of 23 S rRNA, isolated
as well bound to r-proteins in the ribosome, indicates that domain I is more highly structured and
compact than the other domains of the molecule
(Egebjerg et al., 1987, 1990; DoÈring et al., 1991).
Addition of a single, relatively small ribosomal
protein, L24, the only primary binding protein
which binds to domain I, completes the folding of
the domain and initiates assembly of the 50 S subunit (CabezoÂn et al., 1977; Spillman & Nierhaus,
1978; Tumminia et al., 1994). L24 has been footprinted to two sites (Egebjerg et al., 1987), one of
which is adjacent in the primary and secondary
structure to the three-way junction which contains
the loop E motif, and which may therefore play a
role in helping to structure the neighbouring RNA
for interaction with L24.
Loop E and RNA-RNA interactions
In the hairpin ribozyme, it has been shown that
the internal loop containing the Loop E motif must
interact with the loop containing the cleavable
phosphate for catalysis to occur (Burke, 1996;
Burke et al., 1996). A recent model with side-byside interaction of the two internal loops has been
proposed (Earnshaw et al., 1997).
Protection by ribosomal proteins S5 and S12 of
bases in the multi-branch loop at the junction of
the three major domains of 16 S rRNA, including
those belonging to the loop E motif that we have
identi®ed, was observed using base-speci®c probes
(Stern et al., 1988a), but not using hydroxyl radicals
generated with the Fe2‡/EDTA reagent (Powers &
Noller, 1995). Hydroxyl radicals react primarily
with the ribose moieties of the backbone,
suggesting that the base modi®cations were due to
conformational changes induced by binding of
these proteins to other regions of the RNA (notably
the nearby 530 stem-loop region). Thus, it may be
that the loop E motif in the core of the 16 S rRNA
581
Recurrent Motif in Multi-helix Loops in RNA
is involved in RNA-RNA rather than RNA-protein
interactions. In support of this, two distinct RNARNA crosslinks connecting G894 (in the duplex
just above the sheared A A bp of the motif) with
bases U244 in the early-folding 50 domain and
A1468 in the 30 -terminal domain indicate the central role of the core in organizing the domain structure of 16 S rRNA (Wilms et al., 1997).
Crosslinking is also observed between U1345, at
the base of the loop E motif in the 16 S three-way
junction, and unpaired C934, in the same junction
(Wilms et al., 1997). Unpaired bases in the junction,
U1376 and C1378 have been crosslinked directly to
ribosome-bound tRNA (DoÈring et al., 1994). Taken
together with foot-printing of U1381 by bound
tRNA, discussed above, the evidence is strong for
a role for the loop E motif in structuring this threeway junction for RNA-RNA interaction with
tRNA.
Conclusions
Comparative sequence analysis and chemical
probing data show that the loop E motif is a recurrent, autonomous structural element that occurs
not only within RNA ``internal'' but also around
``multi-helix'' loops, which points to crucial and
multiple roles in structuring and organizing the
junctions. Although loop E motifs are often footprinted by proteins, their involvement as RNARNA anchors cannot be excluded, as shown by the
example of the hairpin ribozyme (Butcher & Burke,
1994b; Earnshaw et al., 1997). The speci®city and
the structure of the binding motif, of either protein
or RNA are still unknown.
Methods
Sequence analysis was carried using the COSEQ
program (Massire & Westhof, unpublished). We made
extensive use of the secondary structures of the 16 S,
23 S and group I introns posted on the Web (http://
pundit.colorado.edu:8080/RNA) (Gutell et al., 1993;
Damberger & Gutell, 1994; Gutell, 1994). 5 S rRNA
sequences were obtained from the Berlin database.
Nucleotides are numbered consecutively according to
the Xenopus laevis sequence (Specht et al., 1997). Large
(23 S-like) and small (16 S-like) ribosomal subunit
RNA sequences were obtained from the Antwerp
database and the Ribosomal Database Project (Maidak
et al., 1997; van de Peer et al., 1997a,b). Sequences
belonging to the three main phylogenetic groups
(Woese et al., 1990), eucaryotes (1581 16 S and 64 23 S
rRNA sequences), Archaea (379 16 S and 24 23 S
rRNA sequences), and bacteria (4188 16 S and 180
23 S rRNA sequences) were downloaded and analysed
separately. At positions harbouring likely loop E
motifs, the alignments were checked for insertions or
deletions as an initial screen for the existence of the
motif at a given site. When the absence of insertions
(or deletions) was con®rmed, covariations between the
pairings were analysed using COSEQ. Numbering of
nucleotides and helices for 16 S and 23 S rRNAs are
given according to the Escherichia coli sequences
(Brimacombe, 1991).
Acknowledgements
We are grateful to Christian Massire for help with the
use of the COSEQ program program. N.L. was a Fogerty Senior International Fellow, supported by NIH grants
(1-F06-TW02251-01) and 1R15-GM/OD55898-01 (NBL),
ACS-PRF grant no. 31427-B4 and by a ``Poste Rouge''
from the CNRS. E.W. acknowledges support from the
Institut Universitaire de France.
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Edited by K. Nagai
(Received 17 March 1998; received in revised form 13 July 1998; accepted 29 July 1998)