Gutell 054.jmb.1996.256.0701

J. Mol. Biol. (1996) 256, 701–719
Comprehensive Comparison of Structural
Characteristics in Eukaryotic Cytoplasmic Large
Subunit (23 S-like) Ribosomal RNA
Murray N. Schnare1
, Simon H. Damberger2
, Michael W. Gray1
*
and Robin R. Gutell2,3
Comparative modeling of secondary structure is a proven approach to1
Program in Evolutionary
predicting higher order structural elements in homologous RNA molecules.Biology, Canadian Institute
Here we present the results of a comprehensive comparison of newlyfor Advanced Research
modeled or refined secondary structures for the cytoplasmic large subunitDepartment of Biochemistry
Dalhousie University (23 S-like) rRNA of eukaryotes. This analysis, which covers a broad
phylogenetic spectrum within the eukaryotic lineage, has defined regionsHalifax, Nova Scotia
B3H 4H7, Canada that differ widely in their degree of structural conservation, ranging from
a core of primary sequence and secondary structure that is virtually2
Department of Molecular
invariant, to highly variable regions. New comparative information allows
Cellular and Developmental us to propose structures for many of the variable regions that had not been
Biology, Campus Box 347
modeled before, and rigorously to confirm or refine variable region
University of Colorado
structures previously proposed by us or others. The present analysis also
Boulder, CO 80309, USA
serves to identify phylogenetically informative features of primary and
secondary structure that characterize these models of eukaryotic3
Department of Chemistry
cytoplasmic 23 S-like rRNA. Finally, the work summarized here providesand Biochemistry, Campus
a basis for experimental studies designed both to test further the validityBox 215, University of
of the proposed secondary structures and to explore structure–functionColorado, Boulder, CO 80309
relationships.USA
7 1996 Academic Press Limited
Keywords: 23 S-like rRNA; higher order rRNA structure; comparative
modeling; conserved core; variable regions*Corresponding author
Introduction
The structural complexity of the ribosome
challenges our understanding of its intimate
involvement in protein biosynthesis. As a first step
toward defining the molecular interactions and roles
of the ribosome in translation, it is essential to
acquire detailed structural knowledge about the
constituent parts of this large ribonucleoprotein
particle. Of particular interest are the RNA
components of the ribosome, given that a now-
considerable body of evidence suggests that these
may be directly involved in ribosome function
(Noller, 1991). Indeed, there are indications that
peptidyl transferase activity may reside primarily
in the large subunit (23 S-like) rRNA (Noller et al.,
1992; Noller, 1993). In this context, robust models of
rRNA secondary structure provide a necessary
conceptual basis for the elucidation of structure/
function relationships in the ribosome (Hill et al.,
1990).
Ribosomal RNA sequences have also proven
invaluable for defining phylogenetic relationships
among organisms. Ribosomal RNA-based phylo-
genetic trees have completely changed our per-
spective on the nature and evolutionary
interrelationships of the prokaryotes (Woese,
1987), and have solidified the view that eukary-
otic organelles (mitochondria and plastids) have
an endosymbiotic, (eu)bacterial origin (Yang et al.,
1985; Gray et al., 1984, 1989; Cedergren et al.,
1988). Because the topology of a phylogenetic tree
may be critically dependent on the accuracy of
the sequence alignment employed (Feng & Doolit-
tle, 1987; Olsen & Woese, 1993), trees based on
rRNA sequences are more likely to reflect true
evolutionary relationships when secondary struc-
tures are available to guide the primary sequence
alignment (as in the examples cited above).
Comparative sequence analysis (Fox & Woese,
1975), also known as the phylogenetic approach
(Brimacombe, 1984), is one of the most powerful
0022–2836/96/090701–19 $12.00/0 7 1996 Academic Press Limited

Secondary Structure of Eukaryotic 23 S-like rRNA702
tools currently available for inferring the higher
order structure of large RNA molecules. This
approach is based on the premise that functionally
equivalent regions of an RNA molecule will exhibit
the same secondary and tertiary structure in all
organisms even when the primary sequences are
not identical. Initially, secondary structure elements
were detected by searching a sequence alignment
for compensating base changes in potential helical
regions. In early studies, only standard (canonical)
base-pairs (G-C, A-U) and G·U pairs were con-
sidered. Over the years, the number of available
sequences in RNA databases has steadily increased,
making it possible to apply more sophisticated,
computer-assisted methods to reveal interacting
nucleotide positions (Olsen, 1983; Gutell et al., 1985;
Haselman et al., 1988; Chiu & Kolodziejczak, 1991;
Gutell et al., 1992b; Gautheret et al., 1995). This
approach, which detects positional covariance in an
alignment independent of the ability of the partner
nucleoside residues to form canonical base-pairs in
a helix, has been applied in the latest refinement of
the secondary structures of Escherichia coli 16 S and
23 S rRNAs, to infer novel secondary and tertiary
interactions in these molecules (Gutell et al., 1994;
Gutell, 1995).
The first large subunit rRNA secondary structure
models, proposed in 1981 for E. coli 23 S rRNA
(Glotz et al., 1981; Branlant et al., 1981; Noller et al.,
1981), were based on experimental as well as
comparative data. A secondary structure model for
eukaryotic large subunit rRNA appeared in the
same year, when the sequence of yeast 26 S rRNA
was published (Veldman et al., 1981; Georgiev et al.,
1981). This first eukaryotic 23 S-like rRNA se-
quence, although significantly longer than its E. coli
counterpart, proved to have the potential to form a
core secondary structure very similar to that
proposed for bacterial 23 S rRNA (Veldman et al.,
1981). As additional eukaryotic sequences were
determined, they were compared with each other
and with the available E. coli secondary structure
models, culminating in 1984 in the publication of
secondary structure models for the 23 S-like rRNAs
of yeast (Hogan et al., 1984), rat (Hadjiolov et al.,
1984), mouse (Michot et al., 1984) and Xenopus laevis
(Clark et al., 1984). These studies concluded that a
common secondary structure core is shared by
eukaryotic and bacterial 23 S-like rRNAs, with the
extra length of the eukaryotic sequences restricted
to discrete variable sequence blocks that are
localized to specific regions of the structure.
Concomitant with the rapid increase in the
number and phylogenetic diversity of available
sequences (Gutell et al., 1993), we have proposed
(Gutell & Fox, 1988; see also Wool, 1986) and have
continued to update and improve (Gutell et al., 1990,
1992a, 1993) a compendium of secondary structure
models for 23 S and 23 S-like rRNAs. Over the past
few years we have made substantial progress in (1)
fitting all of the available eukaryotic sequences
(Table 1) to a conserved secondary structure core,
and (2) inferring secondary structures for the
variable regions of eukaryotic large subunit rRNA.
The results of this detailed analysis are summarized
and discussed here.
Results
Overview of secondary structure in eukaryotic
large subunit rRNAs
Complete or nearly complete 23 S-like rRNA
sequences are now available for 42 eukaryotes
spanning a broad spectrum of phylogenetic groups
(Table 1). We have employed comparative methods
to deduce detailed secondary structures for all of
these sequences, and analysis of the resulting
models (available at our World Wide Web site; see
Methods) has defined a shared conserved core
(Figure 1). Particular structural elements discussed
below are designated according to the coordinates
of the corresponding elements in E. coli 23 S rRNA
(rrnB operon), identified on the schematic diagram
in Figure 2. In many cases, comparison of the
proposed eukaryotic structures within and between
phylogenetic groups has led to significant improve-
ments within the core region relative to our
previous proposals (Gutell & Fox, 1988; Gutell et al.,
1990, 1992a, 1993).
Figure 1 also shows all of the comparatively
inferred tertiary interactions as well as a non-
canonical pair, C·A (U·G in Euglena) at E. coli-
equivalent positions 779:785 (Leffers et al., 1987;
Haselman et al., 1989; Gutell & Woese, 1990; Larsen,
1992; Gutell et al., 1994). Several of these proposed
tertiary interactions have since been confirmed by
experimental studies (Ryan & Draper, 1991; Kooi
et al., 1993; Aagaard & Douthwaite, 1994; Rosendahl
et al., 1995). Another non-canonical pair (usually
C·A) is located at positions 1950:1956 (U·G in
Caenorhabditis elegans, G·U in Giardia species, U·U in
Didymium/Physarum).
A number of unique features that characterize a
23 S-like rRNA as eukaryotic, and that distinguish
a eukaryotic 23 S-like rRNA from its prokaryotic
counterparts, can readily be identified within
this core structure. Some of these distinguishing
structural characteristics are summarized in Table 2
(which is not, however, meant to be a comprehen-
sive listing). These features persist in the face of an
almost twofold variation in the length of these
homologous rRNA molecules (Table 1), emphasiz-
ing that size alone is not an adequate criterion for
classifying large subunit rRNAs as ‘‘prokaryotic’’ or
‘‘eukaryotic’’.
In the consensus eukaryotic secondary structure
illustrated in Figure 1, the conserved core is defined
by the presence of a particular nucleotide or
structural element at a given position in at least 90%
of the available sequences (Table 1). In cases where
conservation is <100%, there may be some notable
deviations from the core structure. Some examples
are (again with reference to the E. coli coordinates;
Figure 2): (1) the two hairpins encompassing

Secondary Structure of Eukaryotic 23 S-like rRNA 703
positions 121 to 148 are absent from the Giardia
muris 5.8 S rRNA portion of the structure; (2) the
helix at positions 604 to 624 is truncated to only
three base-pairs in Giardia muris, whereas in most
other eukaryotes it is extended by a few base-pairs
compared to its (eu)bacterial counterpart. Most of
this region remains unstructured in our Euglena
model; (3) the base-pairing at positions 1435 to
1444:1547 to 1557 is not possible in the Crithidia and
Trypanosoma structures; (4) the otherwise conserved
hairpin at positions 1527 to 1544 is absent from the
Giardia models. (Bear in mind that this conservation
analysis is dependent on the sequence space
sampled, so that the results obtained will be skewed
by over- or under-representation of structures from
particular phylogenetic groups.)
For the most part, regions of secondary structure
that are conserved among eukaryotic 23 S-like
rRNAs are superimposable on the same sections of
the (eu)bacterial and archaeal models. Regions of
major structural deviation between the eukaryotic
and prokaryotic core structures are: (1) the section
between positions 76 to 110 is much more highly
structured in the bacterial and archaeal models. In
our eukaryotic models, this region of the 5.8 S rRNA
is drawn as a large loop closed by three base-pairs.
In many eukaryotic sequences this stem can be
extended at its base; this is especially so for Giardia
and related organisms (see Katiyar et al., 1995); (2)
the helix at positions 150 to 176 is extended in
eukaryotes and contains the discontinuity that
separates the 5.8 S and 28 S rRNA molecules, which
together are the structural equivalent of a prokary-
otic 23 S rRNA (Nazar, 1984); (3) the helix at
positions 01860 to 1880 is truncated in all
eukaryotes, a feature shared with archaeal 23 S-like
rRNA secondary structure models (Gutell, 1992)
(archaeal structures are also available at our WWW
site; see Methods).
Eukaryotic large subunit rRNAs range in size
from 2811 nt in Giardia muris to 5185 nt in Homo
sapiens (Table 1). Given that these rRNAs have a
common conserved core of secondary structure, it
follows that this size variation must be accommo-
dated within discrete regions of the structure
outside of the core. The location and size range of
these variable regions are shown in Table 3. Until
recently, given the absence of comparative support
(compensating base changes) for a common
structure, many of these variable regions had not
been modeled. We have now made significant
progress in defining the secondary structure in most
of these variable regions, which often contain
secondary structure that is common to only a
sub-group of eukaryotes (e.g. fungi). The large
G + C-rich variable regions in vertebrate 23 S-like
rRNAs, which remain mostly unmodeled by us,
actually exist as very stable structural features that
are detectable by electron microscopy (Wakeman &
Maden, 1989). Potential secondary structures for
these regions have been proposed by others,
primarily on the basis of thermodynamic consider-
ations (Hassouna et al., 1984; Michot et al., 1984;
Michot & Bachellerie, 1987; Hadjiolov et al., 1984;
Clark et al., 1984; Gonzalez et al., 1985; Gorski et al.,
1987; Leffers & Andersen, 1993).
In the following section we address in turn the
more prominent variable regions found in eukary-
otic large subunit rRNA (Table 3). For this
discussion, we encourage the interested reader to
obtain the complete collection of secondary struc-
ture diagrams from our WWW site (see Methods).
This site also contains partial alignments of variable
regions, which provide the comparative evidence
for newly proposed or refined secondary structures.
Positions 271 to 369
This region begins with an isolated hairpin of
variable length (structure a in Figure 1). The Euglena
sequence has an additional potential helix at the 5'
end of this variable region, which corresponds to
the 3' terminal part of the species 2 component of
the fragmented Euglena gracilis large subunit rRNA
(Schnare & Gray, 1990). In the middle of this
variable region we have identified a phylogeneti-
cally conserved structure (positions 0300 to 340)
that is homologous to the so-called ‘‘3 S rRNA’’ of
Chlamydomonas chloroplasts (Turmel et al., 1991).
This structure can form the same potential tertiary
interactions (317 to 318:333 to 334 and 319:323) that
have been proposed for this region of E. coli 23 S
rRNA (Gutell & Woese, 1990; Gutell et al., 1992b).
The remaining sequences in this variable region
form an irregular helix (271 to 297:341 to 366)
connecting the 3 S-like structure to the rest of the
large subunit rRNA. The overall layout of this
region in secondary structure models is similar in
all eukaryotes; however, the precise details of the
structures do vary among different eukaryotic
groups.
Positions 533 to 560 (545 region)
This is one of the most highly variable regions in
eukaryotic large subunit rRNA, ranging in length
from 8 to 865 nt (see Table 2). It is extremely short in
Giardia species, consisting of a 2 to 12 bp hairpin
with a 3 to 4-base loop. In most other eukaryotes, the
region is hundreds of nucleotides long. In addition
to the complete sequences listed in Table 1, we have
made use of many partial sequences in deriving
structures for the 545 region (displayed in Figure 3).
The partial sequences published by Linares et al.
(1991), Pe´landakis & Solignac (1993), Preparata et al.
(1992), and Fernandes et al. (1993) proved particu-
larly useful in the analysis of this region. Figure 4
provides an example of the comparative data that
we have compiled in support of the variable-region
structural models presented here.
There is some obvious similarity among the
individual structures in the overall layout of our
secondary structure proposals for the 545 region
(Figure 3). Most notably, these structures contain the
H2 helix of Michot & Bachellerie (1987), which

previously (Gutell et al., 1993) we had overlooked.
The H2 helix pairs an internal stretch of nucleotides
to a sequence close to the 3' end of this variable
region (helix E in Figures 3 and 4). We have now
folded the remaining portion of each sequence in
the 545 region into two or three group-specific
structural domains (Figure 3), which draw support
from a large number of compensating base changes.
Generally, this region (see Figure 5) can be
modeled as two hairpin structures, the first having
an internal loop 05 bp removed from the beginning
of the helix. The second hairpin almost always
contains a bulged nucleotide (absent in Giardia and
Entamoeba) on its 3' side, two base-pairs removed
from the beginning of the stem. Most of the length
variation in this 650 region (25 to 127 nt) can be
accounted for by differences in the lengths of these
two helical elements. In some organisms (chordates,
C. elegans and Phytophthora megasperma), the models
contain an additional hairpin near the 5' end of this
variable region.
Although this region is highly variable in
primary sequence, it is usually 037 to 38 nt long
and contains a single 08 to 10 bp hairpin that is
supported by many compensating base changes.
The Aedes albopictus sequence variable region (88 nt
long) has the potential to form an additional
hairpin. Two protist sequences are also considerably
longer than average in this region (Dictyostelium,
61 nt; Euglena, 84 nt), and their potential structures
cannot yet be definitively established. The disconti-
nuity between Euglena rRNA species 5 and 6 is
within this variable domain (Schnare & Gray, 1990).
Although this region is quite divergent in both
length (Table 3) and primary sequence, in most
Table 1. Available eukaryotic 23 S-like rRNA sequences
Organism Length (5.8 S + 28 S, nt) Accession number
A. Animalia
Arthropoda
Aedes albopictus 4262b
L22060
Drosophila melanogaster 4077 M21017
Chordata
Herdmania momus 3721 X53538
Homo sapiens 5185 J01866, M11167
Mus musculus 4869 J01871, X00525
Rattus norvegicusa
4941 X00521
Xenopus borealisa
4289 X59733
Xenopus laevis 4276 K01376, X00136, X59734
Nematoda
Caenorhabditis elegans 3662 X03680
B. Archezoa
Giardia ardeae 2826 X58290
Giardia intestinalis 2837 X52949
Giardia muris 2811 X65063
C. Fungi
Ascomycota
Saccharomyces carlsbergensisa
3551c
J01352, V01285, V01325
Saccharomyces cerevisiae 3550 J01355, K01048
Schizosaccharomyces japonicus 3578 Z32848
Schizosaccharomyces pombe 3662 J01359, Z19136
Basidiomycota
Cryptococcus neoformans 3544 L14067, L14068
Deuteromycota
Candida albicans 3513 X71088, X70659, L28817
Zygomycota
Mucor racemosus 3627c
M26190
Unknown
Pneumocystis carinii 3503 M86760
D. Plantae
Angiospermophyta (flowering plants)
Arabidopsis thaliana 3539 X52320
Brassica napus 3542 D10840
Citrus limona
3557c
X05910
Fragaria ananassa 3541 X15589, X58118
Lycopersicon esculentum 3544 X52265, X13557
Oryza sativa 3541 M16845, M11585
Sinapis alba 3544 X15915, X57137
continued

Table 1. continued
Organism Length (5.8 S + 28 S, nt) Accession number
E. Protista
Acrasiomycota (cellular slime molds)
Dictyostelium discoideum Incomplete X00601
Apicomplexa
Theileria parva 3514c
L26332, L28036, L28998
Toxoplasma gondii 3625 X75429, X75430, X75453
Chlorophyta (unicellular green algae)
Chlorella ellipsoidea 3513 D17810, D13340
Ciliophora (ciliates)
Tetrahymena pyriformisd
3497b
M10752, X54004
Tetrahymena thermophila 3497 X54512
Dinoflagellata (dinoflagellates)
Prorocentrum micans 3567b
M14649, X16108
Euglenophyta (euglenoid flagellates)
Euglena gracilis 4052 X53361
Myxomycota (plasmodial slime molds)
Didymium iridis 3857 X60210
Physarum polycephalum 3943 V01159
Oomycota
Phytophthora megasperma 3860 X75631, X75632
Rhizopoda (amastigote amoebas)
Entamoeba histolytica 3674b,c
X65163
Zoomastigina (zooflagellates)
Crithidia fasciculata 4077 Y00055
Trypanosoma brucei 4188 X05682, X14553, X04986
Trypanosoma cruzia
4334 L22334, X54476
Eukaryotic organisms are classified according to the scheme of Margulis & Schwartz (1988). See
Cavalier-Smith (1987) for a definition of kingdom Archezoa. Original references for most of these sequences
are listed by Gutell et al. (1992a, 1993). New references are: Aedes albopictus (Kjer et al., 1994),
Schizosaccharomyces japonicus (Naehring et al., 1995), Schizosaccharomyces pombe (Lapeyre et al., 1993),
Cryptococcus neoformans (Fan et al., 1994), Candida albicans (Mercure et al., 1993; Srikantha et al., 1994), Theileria
parva (Kibe et al., 1994; Bishop et al., 1995), Toxoplasma gondii (Y. Ding, S. M. Fisenne & B. J. Luft, unpublished),
Chlorella ellipsoidea (Aimi et al., 1992, 1994), Phytophthora megasperma (Van der Auwera et al., 1994) and
Trypanosoma cruzi (Galvan et al., 1991; E. Go´mez, S. Martinez-Calvillo & R. Hernandez, unpublished).
a
Secondary structure diagrams for these sequences are not available at our WWW site. However, these
structures are virtually identical to those of closely related species.
b
Actual size of the mature rRNA from these species is expected to be somewhat less due to excision of
additional internal transcribed spacers (not fully characterized) from the 28 S rRNA transcript.
c
Sizes of these incompletely sequenced RNAs were estimated by comparison with other sequences.
cases it has the potential to form a simple hairpin
structure. In some (eu)bacterial and plastid 23 S
rRNAs, this region contains a discontinuity (see
Trust et al., 1994; Turmel et al., 1991) that results
from excision of an internal transcribed spacer;
however, none of the fragmented eukaryotic
23 S-like rRNAs has a discontinuity at this position
(see Schnare et al., 1990).
In the majority of eukaryotic sequences, this
region is conserved in size and structure, and
contains a hairpin having a 6 bp helix and (usually)
a 7 nt loop. However, there are a few exceptions. In
several Giardia species both the helix and loop are
smaller in size, whereas the loop is significantly
larger in Euglena, Entamoeba, Crithidia and Try-
panosoma species.
In many eukaryotes, the sequences at the two
ends of this variable region interact to form a 2 bp
helix; however, we have uncovered comparative
evidence indicating that a 4 bp helix is present in
some sequences. The remaining sequence in this
region forms two hairpin structures, with a
discontinuity in the Euglena and Tetrahymena
23 S-like rRNAs located in the loop of the second
hairpin. The first hairpin usually contains 3 to 6 bp,
whereas most of the variation in sequence and
length (Table 3) is confined to the second hairpin.
This region contains a single short hairpin in
plants, fungi and most protists. Animals, Entamoeba
and Didymium/Physarum have an additional hair-
pin that accounts for the size variation (see Table 2),
whereas Crithidia and Trypanosoma species have a
discontinuity at this site. In the majority of the
eukaryotic structures, the first hairpin is capped
with a GNRA tetraloop.
In this region, many of the available sequences
have an 0170 +/−30 nt portion that can be
modeled as a basically similar element in a wide

range of eukaryotes (Figure 6). In most cases, the
ends of this variable region interact to form an 022
bp helix that contains an internal loop after
base-pair 14 (one bulged nucleotide on the 5' side
and six bulged nucleotides on the 3' side of the
helix). There are also two bulged nucleotides on the
3' side of the helix, nine bp removed from the
beginning of the helix, with a mismatched C·A
representing the third base-pair in all eukaryotes.
The remaining part of this variable region is
composed of two helices of differing length (see
Figure 6). In many of the structures, one or both of
these helices are capped with UUCG, CUUG or
GNRA tetraloops. In a few organisms (i.e. animals,
Crithidia and Trypanosoma species) whose 23 S-like
rRNA contains hundreds of additional nucleotides
(a)
Figure 1(a)

(b)
Figure 1. Consensus secondary structure of eukaryotic 23 S-like rRNA ((a) 5'-half; (b) 3'-half). The consensus of 42
sequences (see Table 1) is superimposed onto an large subunit rRNA secondary structure diagram. Positions that are
conserved at an identity level >90% are shown with letters. Bold uppercase letters denote conserved sites at which a
particular nucleotide occurs, whereas lowercase letters indicate conserved positions occupied by two different
nucleotides (r = A or G; y = U or C; m = A or C; k = G or U; s = G or C; and w = A or U). Open circles designate positions
that are present in 90% of the sequences but that do not show a signiﬁcant degree of conservation at the primary
sequence level. Secondary structure helices that are more variable than those indicated with open circles but
nevertheless are generally alignable are outlined in schematic form with continuous lines. Regions that vary greatly
in size (variable regions) are depicted as arcs or loops, with numbers indicating the size variance. Base-pairing is
indicated as follows: standard canonical pairs by lines (C-G, G-C, A-U, U-A); wobble G·U pairs by dots (G·U); A·G
pairs by open circles (A)G); other non-canonical pairs by ﬁlled circles (e.g. C,A). Tertiary interactions are shown
connected by continuous lines (where there is strong comparative support) and dotted lines (where comparative support
is moderate).

Figure2.CoordinatesmapofE.coli23SrRNA(left,5'-half;right,3'-half).AschematiclinedrawingofthesecondarystructuremodelfortheE.coli23SrRNA
(Gutelletal.,1993,1994)isshown,withbase-pairingandtertiaryinteractionsthatlinkthe5'-and3'-halvesofthemoleculeindicatedasinFigure1.Every50th
positionisnumberedandevery10thpositionisidentiﬁedwithatickmark(=).TheapproximatepositionsofthevariableregionslistedinTable3areboxed.The
threeregionsshownindetailinFigures3,5and6(533to560,637to653and1707to1751,respectively)areshaded.

Table 2. Distinguishing structural features in eukaryotic 23 S-like rRNAs
Feature Positionsa
(Eu)bacteria Archaea Eukaryotes
Canonical base-pair 823:834 + + − (U·U)
1709:1749 + + − (C·A)
2550:2558 + + − (usually
A·C)
Insertionb
after: 740 − + +
742 − + +
1378 − + +
1564 − + +
1845 − − +
02186 − − +
2257 − + +
Deletionb
at: 739 − + +
896 − + +
957 − + +
995 − + +
2402 − + +
a
E. coli coordinates (see Figure 2).
b
Single nucleotide.
in this region (see Table 3), this part of the structure
is uncertain. The entire variable region is truncated
and is represented by a short hairpin in Entamoeba
(18 bp) and Giardia species (08 to 11 bp).
This variable region ranges in size from 6 to 65 nt
(Table 3). The structures for this region have strong
comparative support and are usually represented
by a hairpin of 2 to 3 bp closed by a common
tetraloop sequence (UUCG, CUUG or GNRA). This
region is reduced to 1 bp in Giardia species and is
extended in some animal species. The helix is also
extended in Euglena, with the loop region contain-
ing the discontinuity that separates rRNA species 8
and 9 (Schnare & Gray, 1990).
This region ranges in size from 14 to 287 nt but
in most cases is 070 to 80 nt long. Its 5' and 3' ends
form an 09 bp helix containing an internal loop
(usually ﬁve unpaired nucleotides on the 5' side and
Table 3. Variable regions in eukaryotic 23 S-like rRNA
Number of nucleotides
Coordinatesa
E. coli Animalia Archezoa Fungi Plantae Protista
81–105 25 20–21 13–20 22 22 18–30
131–148 18 21–23 0–12 21–23 23–24 18–34
271–369 99 158–167 107–114 152–160 149–151 142–215
533–560 28 233–865 8–29 207–313 214–227 205–404
637–653 17 95–127 25–43 66–75 64–66 53–103
845–847 3 5–29 4–6 6–7 7–8 4–26
929–932 4 36–88 37 37–39 38 35–84
1023–1026 4 9–12 1–2 10 11 8–13
1164–1185 22 39–199 14 25–30 27 24–61
1276–1294 19 19–20 12–15 19 19 19–48
1355–1376 22 46–97 39–43 49–51 41–45 41–147
1413–1419 7 41–83 19–22 25 24 23–58
1473–1518 46 46–49 45 46–51 46–47 46–70
1527–1544 18 18–20 0 18–19 19–20 19
1579–1589 11 14–18 7–9 15–28 15–16 8–29
1707–1751 45 108–718 20–28 140–204 167–169 46–345
2127–2161 35 9–65 6 8–16 8–12 8–23
2200–2223 24 83–287 14 70–76 73–78 64–255
2400–2402 3 4 3–4 4–5 3–4 3–77
2626–2629 4 3–14 0–2 1–4 1 3–121
2789–2810 22 130–235 20–42 118–148 127–135 81–186
2832–2885 54 69–77 58–70 73–77 76–77 69–111
In the context of this Table, ‘‘variable regions’’ are deﬁned as those portions of the sequence
within which the length varies by more than 10 nt among the compared species. The boundaries
of some of these regions were chosen to correspond to the ends of helical regions and therefore
overlap parts of the conserved core. Note that there is also length variation at the 3'-5.8 S/5'-28 S
junction (see Figure 1); however, it is possible that this variation could be the result of inaccurate
mapping of the ends of the mature rRNAs.
a
Positions in E. coli 23 S rRNA.

Figure 3. The 545 gallery. A collection of phylogenetically diverse secondary structure diagrams is shown for variable
regions corresponding to positions 533 to 560 (545 region) in E. coli 23 S rRNA (see inset and Figure 2). Representative
models are presented for the ﬁve major phylogenetic groups (Plantae, Archezoa, Fungi, Protista, and Animalia). Primary
sequence in large unstructured regions is not displayed; instead, these regions are denoted by an arc, with numerals
indicating number of nucleotides. The majority of the base-pairs shown are supported by at least one compensatory
change (see Figure 4). Helices discussed in the text and in Figure 4 are indicated by uppercase letters (A to K) on the
C. neoformans secondary structure.

two unpaired nucleotides on the 3' side). The
remaining internal portion of this variable region is
typically found as two hairpins, with the second
one usually flanked by AA on its 5' side and by GA
on its 3' side. An additional helical region situated
between the two hairpins described above is
probable for the Euglena, Crithidia and Trypanosoma
sequences. We have also derived a unique structure
for this variable region in Didymium and Physarum;
this was confirmed by comparison with the
sequence from Physarum flavicomum (Vader et al.,
1994). In Giardia species this variable region is
represented by a 4 bp helix with a 6 nt loop.
This variable region is usually only a few
nucleotides long (Table 3) and contains a discontinu-
ity in Euglena, Crithidia and Trypanosoma species.
We have identified group-specific structures for
Didymium/Physarum (27 nt) and Crithidia/Try-
panosoma (0120 nt).
This region is highly variable in size (Table 3),
primary sequence and secondary structure. In most
eukaryotic 23 S-like rRNAs we have inferred a helix
formed by interaction of sequences near the 5' and
3' termini of this variable domain. In Giardia species,
the entire region is reduced to a single hairpin of 6
to 8 bp. In plants and fungi, comparative evidence
supports lineage-specific structures, each having
two internal hairpins. Among vertebrates, the
sequence in this region is highly conserved; in
Xenopus, however, an apparent deletion has
removed part of the sequence that in other verte-
brates contributes to phylogenetically established
secondary structure. In most other sequences, we
have identified either one or two internal hairpins in
Figure 4. Comparative support for secondary structure in the 545 region of fungal eukaryotic 23 S-like rRNAs (see
Figure 3). Uppercase letters denote helices (see C. neoformans structure in Figure 3), with parentheses enclosing the
single-strand loop of a hairpin structure. Numbers refer to positions that pair in the secondary structure (e.g. A1 at
the beginning of the sequence pairs with A1 at the end of the sequence, etc.) and that display compensating base changes
that support the inferred structure. A plus sign (+) marks a position at which no compensating base changes occur,
whereas a caret (g) denotes a position that displays non-canonical base-pairs that are consistent with canonical pairs
found at the same position (e.g. the second pair in helix A, which is occupied by G-C, G·U or A-U). Organism names
are abbreviated as follows: Pca, Pneumocystis carinii; Sja, Schizosaccharomyces japonicus; Spo, Schizosaccharomyces pombe;
Sce, Saccharomyces cerevisiae; Cne, Cryptococcus neoformans; Cal, Candida albicans; Mra, Mucor racemosus.

regions corresponding to positions 0637 to 653 (650 region) in E. coli 23 S rRNA (see inset and Figure 2). For details
of the structural representation, see Figure 3.
this region. In Euglena the 3' end of rRNA species
11 is paired to a sequence near the 5' end of species
12 to form an additional helix that is homologous to
a hairpin identiﬁed in the 070 nt small rRNA in
Crithidia and Trypanosoma species (see Schnare &
Gray, 1990).
In this region, most of the sequences are 075 nt
in length and conform to the structure that we had
previously proposed for Saccharomyces cerevisiae
cytoplasmic 23 S-like rRNA (Gutell et al., 1993). This
structure is similar to the E. coli model, with an
extension of a helix corresponding to positions 2852
to 2865. The length variation in this region (Table 3)
can mostly be accounted for by the shorter
sequences in Giardia species and the longer
sequences in Euglena and Crithidia/Trypanosoma
species. These length variations result in deviations
from the yeast secondary structure model in this
region.

regions corresponding to positions 1707 to 1751 (1707 region) in E. coli 23 S rRNA (see inset and Figure 2). For details
of the structural representation, see Figure 3.

Discussion
More than a decade of comparative sequence
analysis has culminated here in the proposal of
complete or nearly complete secondary structures
for all eukaryotic large subunit rRNA sequences
available at this time. These newly modeled or
refined structures are accessible electronically via
the WWW (see Methods). The validity of the
proposed secondary structures was evaluated
according to two criteria. First, new sequences
added to the database can be viewed as tests of our
current models. Upon re-analysis after such
additions, revisions are no longer required to the
core structure, nor indeed to most of the variable
regions. Thus, these structures are now refined
to the point where they consistently pass the
challenge of newly determined sequences. When
archaeal, (eu)bacterial and organellar structures
(also available at our WWW site) are also taken
into consideration, we find compensating base
changes at almost every proposed base-pair within
the conserved core. The comprehensive size and
scope of the database, especially among plants and
fungi, has also allowed us to discern a large number
of compensating base changes in many of the
variable regions. Thus, in a substantial number of
cases there is strong comparative evidence in
support of the new structures proposed here (e.g.
Figure 4).
Secondly, we have evaluated our secondary
structure models in relation to published exper-
imental data. A large body of experimental
evidence supports the proposed E. coli structure
(Hill et al., 1990), and much of this evidence is also
applicable to the eukaryotic core. Our structures are
also supported by experimental data derived
specifically from eukaryotic systems, as outlined
below.
The results of experiments designed to probe the
secondary structure of free 5.8 S rRNA in solution
(reviewed by Nazar, 1984) have prompted sec-
ondary structure models that are not supported by
broad phylogenetic comparisons (MacKay et al.,
1982), even when interactions between 5.8 S and
28 S rRNA are taken into consideration (Olsen &
Sogin, 1982). On the other hand, it has been
demonstrated in several systems (see Nazar, 1984)
that the conformation of ribosome-associated 5.8 S
rRNA differs substantially from that of 5.8 S rRNA
free in solution. In the case of 5.8 S rRNA structure
probed in 60 S subunits or in intact ribosomes (Lo
& Nazar, 1981, 1982; Wildeman & Nazar, 1982; Liu
et al., 1983; Lo et al., 1987; Holmberg et al., 1994a,b),
the available experimental data are entirely consist-
ent with our proposed model, but are incompatible
with previous ‘‘universal’’ models (e.g. see Michot
et al., 1984; Vaughn et al., 1984). In experiments in
which the conformation of regions of 28 S rRNA
was probed in the absence of ribosomal proteins
(Qu et al., 1983; Stebbins-Boaz & Gerbi, 1991; Ajuh
& Maden, 1994), the results suggest a structure that
is generally compatible with our secondary struc-
ture model. In some of the regions in which the
structure of free Xenopus 28 S rRNA deviates from
our model, it has been demonstrated that confor-
mational changes in the RNA occur in 60 S subunits
and 80 S monosomes; these changes give rise to 28 S
rRNA probing data that are much more consistent
with our model (Stebbins-Boaz & Gerbi, 1991).
The conformation of 23 S-like rRNA within 60 S
subunits has been probed with kethoxal in yeast
(Hogan et al., 1984) and with dimethylsulphate
and 1-cyclohexyl-3-(morpholinoethyl)carbodiimide
metho-p-toluene sulphonate (CMCT) in mouse
(Holmberg et al., 1994a). Most but not all of the
data from those studies are consistent with the
secondary structure models that we propose for the
cytoplasmic 23 S-like rRNAs of these organisms. In
a few instances the experimental data conflict with
our models, most likely reflecting the fact that the
ribosome is not a static structure; thus, some of the
interactions we have inferred by comparative
analysis may only be present during specific stages
of ribosome biogenesis and/or protein biosynthesis
(Hogan et al., 1984; Holmberg et al., 1994a).
Many publications of large subunit rRNA
sequences are accompanied by proposed secondary
structures, and several papers have presented
phylogenetic analyses of potential structure for
particular regions of 23 S-like rRNA (Michot &
Bachellerie, 1987; Lenaers et al., 1988; Bachellerie &
Michot, 1989; de Lanversin & Jacq, 1989; Michot
et al., 1990; Linares et al., 1991; Rousset et al., 1991).
Although a detailed comparison is beyond the
scope of this paper, it is fair to say that our
structures are not identical to any other pub-
lished versions. However, many features of our
structures can be found in one or more of the other
published eukaryotic secondary structure models.
We note that the secondary structure proposals of
Michot, Bachellerie and co-workers (Hassouna
et al., 1984; Michot et al., 1984, 1990; Michot &
Bachellerie, 1987; Bachellerie & Michot, 1989) have
held up remarkably well considering the limited
number of sequences available at the time the
structures were initially published. The study
summarized here, based on a much more compre-
hensive analysis than previously possible, has been
able to provide strong confirmation for a number of
published variable-region structures that could only
be considered tentative when they were originally
proposed.
At this juncture, within the constraints of the
database of available 23 S-like rRNA sequences, we
are confident that the secondary structures pre-
sented and discussed here are highly refined. We
anticipate that most of these models will be
refractory to major revision as additional rRNA
sequences are determined and analyzed; neverthe-
less, we do anticipate minor refinements in some of
the variable regions. We expect that future revisions
will, for the most part, be restricted to the protist
models, in particular those for which closely related
sequences are not yet available. We therefore
encourage users of these models to consult our

WWW site regularly to ensure that the most recent
version of any particular structure is being used.
Within the core structure of eukaryotic large
subunit rRNAs, there are many stretches of highly
conserved primary sequence. The majority of
known functional sites in 23 S-like rRNA map to
these regions, including the peptidyl transferase
center (parts of domains IV and V), the GTPase-as-
sociated center (around position 1067 in domain II),
and the site of interaction with elongation factors
(around position 2660 in domain VI) (reviewed by
Raue´ et al., 1988; Hill et al., 1990; Noller, 1991, 1993;
see also Leviev et al., 1995; Rosendahl et al., 1995).
One of the most highly conserved regions of
primary sequence in eukaryotic 23 S-like rRNA,
positions 562 to 589, has not yet been implicated
in ribosome function. This region contains two
overlapping 13 nt sequence blocks that may interact
with small nucleolar RNAs U18 and U21, which are
thought to be involved in some aspect of ribosome
biogenesis (Prislei et al., 1993; Qu et al., 1994;
Bachellerie et al., 1995). Several other small
nucleolar RNAs are also complementary to the
highly conserved regions of eukaryotic large
subunit rRNA identified by comparative analysis
(Bachellerie et al., 1995; Qu et al., 1995).
Regions that are most highly variable in size and
structure are exposed on the surface of the ribosome
(Han et al., 1994), where they are less likely to
interfere with the assembly and function of the
conserved core. It has been suggested (Frank et al.,
1990; Han et al., 1994) that the larger variable
regions may represent at least part of the eukaryotic
lobes identified by electron microscopy of eukary-
otic ribosomes. In contradistinction to the conserved
core, it seems unlikely that any of the variable
regions would perform any phylogenetically con-
served functions. In fact, some of the variable
regions have been experimentally altered in an
effort to evaluate their functional importance. In
yeast, artificial extension of the hairpin at position
0271 (structure a, Figure 1) has no effect on
ribosome production or function (Musters et al.,
1989); as well, the hairpin that begins at position
01370 is dispensible (Musters et al., 1991). When
transformants of Tetrahymena thermophila that con-
tain a 119 bp insert in the 02800 region of the
rDNA are cultured, they grow normally and
produce 26 S rRNA containing the insert (Sweeney
& Yao, 1989). On the other hand, the variable region
at positions 1707 to 1751 has an essential role in
either pre-rRNA processing or stabilization of the
mature 23 S-like rRNA in T. thermophila (Sweeney
et al., 1994). It is within this region of human
23 S-like rRNA that small nucleolar RNA E2 is
thought to interact (Rimoldi et al., 1993).
The models presented here should be viewed as
minimal structures because, by definition, the
comparative approach can only detect interactions
in parts of the sequence that actually vary. In
addition, the comparative method only detects the
component of rRNA structure that is mediated by
base:base interactions, providing no information
about structure contributed by stacking of helices,
modified nucleosides, interactions involving the
phosphate-sugar backbone, etc. Additional tertiary
interactions, not yet characterized in detail, have
recently been inferred in a synthetic oligonucleotide
corresponding to the GTPase center of E. coli 23 S
rRNA (Laing & Draper, 1994). These interactions are
stabilized by magnesium and ammonium ions
(Wang et al., 1993; Laing et al., 1994). Presumably the
same interactions exist in eukaryotic 23 S-like
rRNA, because in yeast the GTPase center can be
replaced by its E. coli counterpart without loss of
ribosome function (Musters et al., 1991).
One of the long-term goals of the research
summarized here is to associate rRNA structure
with phylogeny. In other words, we would like to be
able to infer phylogenetic relationships directly
from higher order structure. In this paper, we have
pointed out that within eukaryotic large subunit
rRNAs, there is a range of group-specific structural
features that can potentially be used as a biological
key (see Gutell, 1992). We anticipate that some new
sequences will be compatible with our existing
structures, thereby establishing their phylogenetic
positions. On the other hand, there are still many
phylogenetic groups for which no 23 S-like rRNA
sequences are yet available; thus, we expect to
identify additional, phylogenetically unique sec-
ondary structural elements as further sequences
appear. For example, in the recently determined
partial 23 S-like rRNA sequences from Rotaliella
elatiana (Pawlowski et al., 1994a) and related
Foraminifera (Pawlowski et al., 1994b), there are
several deviations from the conserved core pre-
sented here, most notably in the 730 to 740 region.
These sequences also contain many large insertions
in unexpected places. These foraminiferan 28 S
rRNAs are split into at least four pieces, and
Pawlowski et al. (1994b) propose that some of the
large novel inserts may be removed during the
fragmentation process. In this regard, we would
point out that although one insert is listed in the R.
elatiana annotation, accession number X78521, as an
internal transcribed spacer (after E. coli position
1928), this sequence is actually a group I intron
(S. H. Damberger & M. N. Schnare, unpublished
analysis).
Methods
The alignment editor AE2 (developed by T. Macke; see
Larsen et al., 1993) was used to align sequences manually
on the basis of primary sequence similarity and
previously established eukaryotic secondary structure
features (Gutell & Fox, 1988). Newly identified structural
elements in E. coli 23 S rRNA (see Gutell et al. (1994) for
the most recent version) were also taken into consider-
ation. The aligned database was then subjected to an
iterative process of comparative sequence analysis
(Gutell, 1992; Gutell et al., 1985, 1992b, 1994), as follows:
(1) searches were conducted for compensating base
changes, initially by eye and then with computer
programs as the latter became available; (2) this
information was used to infer additional secondary

structural features, which in turn helped to improve/
refine the sequence alignment; (3) the revised alignment
was re-analyzed and the entire process was repeated until
the proposed structures were entirely compatible with
the alignment; (4) as new sequences became available,
they were aligned against their closest relatives and used
to (i) check the robustness of the existing secondary
structure, (ii) further refine the alignment, and (iii) search
for new structure.
In several of the variable regions, we were unable to
find a common structure for all known eukaryotic
sequences. Therefore, we searched for common structure
among members of smaller phylogenetic groupings (e.g.
fungi, plants). We also carried out literature and database
searches for related partial sequences, some of which
were useful for revising and/or extending the secondary
structure models in certain of the variable regions.
Group-specific structures were proposed for those
variable-region sequences that displayed convincing
homology as well as compensating base changes.
Secondary structure diagrams were generated with the
computer program XRNA (developed by B. Weiser and
H. Noller, University of California, Santa Cruz, and
recently released on the Internet; ftp://fangio.ucsc.edu/
pub/XRNA). The complete and nearly complete eukary-
otic large subunit rRNA sequences that were used in our
analysis are listed in Table 1.
Continually updated 23 S-like rRNA structures are
freely available on-line from an electronic database
maintained at the University of Colorado. Access to these
23 S-like rRNA structures is by anonymous ftp or through
the World Wide Web (WWW). These computer files are
distributed in PostScript format only.
The ftp address and directory are:
pundit.colorado.edu (128.138.212.53)
/pub/RNA/23 S
The WWW address is:
URL:http://pundit.colorado.edu:8080/RNA/23 S/23s.html
Secondary structures in hard-copy format are available
to those unable to access on-line data. Requests for or
correspondence about hard-copy structures should be
sent to M.W.G. Inquiries regarding on-line access to
23 S-like rRNA structures should be sent to R.R.G.
M.W.G. R.R.G.
Tel: (902)494-2521 (303)492-8595
FAX: (902)494-1355 (303)492-7744
E-mail: mgray@ac.dal.ca Robin.Gutell@colorado.edu
Acknowledgements
We gratefully acknowledge the computer programming
expertise of Bryn Weiser (XRNA) and Tom Macke (AE2
Sequence Editor) and the assistance and advice of David
F. Spencer. This work was supported by an operating
grant (MT-11212) from the Medical Research Council of
Canada to M.W.G. and by NIH grant GM48207 to R.R.G.,
who also acknowledges a generous donation of computer
equipment from SUN Microsystems. We thank the W.M.
Keck Foundation for their strong support of RNA science
on the Boulder campus, and the Canadian Institute for
Advanced Research (CIAR) for continuing financial
support in the production and distribution of our hard
copy compendium of 23 S-like rRNA secondary struc-
tures. M.W.G. is a Fellow and R.R.G. is an Associate in the
Program in Evolutionary Biology of the CIAR.
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Edited by D. E. Draper
(Received 12 September 1995; accepted 15 November 1995)

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