The Codex of Business Writing Software for Real-World Solutions 2.pptx
Gutell 084.jmb.2002.323.0035
1. Distribution of rRNA Introns in the Three-dimensional
Structure of the Ribosome
Scott A. Jackson1
, Jamie J. Cannone2
, Jung C. Lee3
, Robin R. Gutell2
*
and Sarah A. Woodson4
*
1
Department of Chemistry
and Biochemistry
University of Maryland
College Park, MD 20497-2021
USA
2
The Institute for Cellular
and Molecular Biology
and Section of Integrative
Biology, The University of
Texas at Austin, TX 78712
USA
3
Division of Medicinal
Chemistry, College of
Pharmacy, The University of
Texas at Austin, TX 78712
USA
4
T. C. Jenkins Department
of Biophysics, Johns Hopkins
University, 3400 N. Charles
Street, Baltimore
MD 21218-4118, USA
More than 1200 introns have been documented at over 150 unique sites in
the small and large subunit ribosomal RNA genes (as of February 2002).
Nearly all of these introns are assigned to one of four main types: group
I, group II, archaeal and spliceosomal. This sequence information has
been organized into a relational database that is accessible through the
Comparative RNA Web Site (http://www.rna.icmb.utexas.edu/) While
the rRNA introns are distributed across the entire tree of life, the
majority of introns occur within a few phylogenetic groups. We analyzed
the distributions of rRNA introns within the three-dimensional structures
of the 30 S and 50 S ribosomes. Most sites in rRNA genes that contain
introns contain only one type of intron. While the intron insertion sites
occur at many different coordinates, the majority are clustered near
conserved residues that form tRNA binding sites and the subunit
interface. Contrary to our expectations, many of these positions are not
accessible to solvent in the mature ribosome. The correlation between the
frequency of intron insertions and proximity of the insertion site to
functionally important residues suggests an association between intron
evolution and rRNA function.
q 2002 Elsevier Science Ltd. All rights reserved
Keywords: group I/II introns; ribosomal RNA; intron transposition; reverse
splicing; sequence database*Corresponding authors
Introduction
Introns in ribosomal RNA genes are found pre-
dominantly found within conserved sequences
near tRNA and mRNA binding sites, suggesting a
possible link between intron evolution and rRNA
function.1 – 3
Examples of every major intron class
have been identified in rRNA genes.4,5
These
include group I and group II introns,6,7
tRNA-like
introns in archaeal genomes,8
a newly defined
family of “spliceosomal” introns in eukaryotic
nuclear rDNA with splice sites that resemble the
conserved splice site sequences of nuclear pre-
mRNA introns,9
and a small number of introns
that cannot be assigned to one of these four groups.
The sporadic appearance of group I and group II
introns among the rRNA genes of organisms from
all three phylogenetic kingdoms points to a
complex evolutionary past.10
Although examples
of introns that have descended through ancient
lineages are known,11 – 14
the appearance of similar
introns in different genes or unrelated organisms
suggests that they were inserted into host genomes
many times during their evolution.15 – 19
Conse-
quently, the distribution of known rRNA introns
is the product of multiple insertions and selective
losses.20
Since ribosomal RNAs are excellent chrono-
meters by which to measure phylogenetic
relationships,21
many laboratories are determining
rRNA sequences from organisms spanning the
entire tree of life. Consequently, GenBank contains
nearly 10,000 complete 16 S and 23 S (and 16 S-like
and 23 S-like) sequences.22
From this diverse
collection of rRNA sequences, approximately 1200
introns have been identified, sequenced, and
0022-2836/02/$ - see front matter q 2002 Elsevier Science Ltd. All rights reserved
E-mail addresses of the corresponding authors:
swoodson@jhu.edu; robin.gutell@mail.utexas.edu
Abbreviations used: ASA, accessible surface area;
CRW, Comparative RNA Web.
doi:10.1016/S0022-2836(02)00895-1 available online at http://www.idealibrary.com on
Bw
J. Mol. Biol. (2002) 323, 35–52
4. organized into a relational database.5
Since the
rRNA sequences flanking the introns are con-
served, the intron/exon boundaries have been
mapped unambiguously. The collections of rRNA
and intron sequences are sufficiently large to inde-
pendently determine the phylogenetic relation-
ships of the introns and the host organisms.
The prevalence of introns in regions of the 16 S
and 23 S rRNAs that bind tRNAs and elongation
factors sparked the suggestion in 1993 that this dis-
tribution arises from reverse splicing into rRNA
sites that are solvent-accessible in ribosomes.23
Other experiments showed that the efficiency of
pre-rRNA splicing depends on interactions
between the intron and the surrounding rRNA.24–27
Both of these results suggest that introns are more
frequent in certain regions of rRNA genes than
others, due to differences in the conformations of
the mature rRNAs at those sites.
Here, we revisit this question in light of the sig-
nificant increase in the collection of rRNA introns
and the recent high-resolution structures of the
ribosome.28 – 31
We have determined that intron-
containing sites are strongly clustered around pos-
itions of the rRNA that interact with tRNAs and
mRNA, but these positions are not more accessible
to solvent in the mature ribosome than average.
Although the splicing mechanism of each intron
class is distinct, most types of introns cluster
within the same regions of the rRNA, suggesting
that the movement and retention of introns is
driven by structural features in the rRNA. The
implications of these findings for mechanisms of
intron mobility and retention are discussed.
Results
A total of 1253 rRNA sequences containing
introns were retrieved from GenBank as of
February 2002, representing organisms from all
major phylogenetic groups. The positions of the
introns within the rRNA gene were determined
from alignments of the mature rRNAs, as pre-
viously described,5
and numbered according to
the sequences of the Escherichia coli 16 S and 23 S
rRNAs.32
Sequence entries were sorted according
to the position of the intron in the 16 S or 23 S
rRNA, and the number of rRNA sequences con-
taining an intron at each position was noted (Table
1). The introns were also classified as group I6,33
or
group II7
families, tRNA-like archaeal introns,26
or
nuclear spliceosomal introns,9,34
based on their
conformity to consensus secondary structures
and conserved sequences. Group I and group II
introns were further subdivided into structural
families.6,7,33
The database of rRNA introns is continually
updated as new rRNA sequences are deposited in
GenBank.5
Hence, the precise statistics reported
here are expected to change as the number of
available intron-containing sequences increases.
However, the number of intron-containing pos-
itions in the rRNA is increasing more slowly than
Table 1 Continued
Intron typea
Cell locationb
Positionc
No. intronsd
I II A S U C M N
2004 1 – – – – 1 – – 1
2059 4 – 4 – – – – 4 –
2066 6 6 – – – – – – 6
2067 1 1 – – – – – 1 –
2256 1 1 – – – – – 1 –
2262 5 5 – – – – 5 – –
2449 48 48 – – – – 7 30 11
2451 1 – 1 – – – – 1 –
2455 1 – 1 – – – – 1 –
2499 1 1 – – – – – 1 –
2500 32 32 – – – – 10 22 –
2504 5 5 – – – – – 5 –
2509 2 – 2 – – – – 2 –
2552 1 – – 1 – – – – 1
2563 7 7 – – – – – – 7
2585 2 2 – – – – – 2 –
2593 16 16 – – – – 13 3 –
2596 3 3 – – – – 3 – –
2601 2 – – 2 – – – – 2
2610 1 – 1 – – – – 1 –
Total 335 269 13 21 29 3 76 105 154
a
Intron types are classified as I, group I; II, group II; A, archaeal; S, spliceosomal; U, unknown.
b
Cell compartment of rRNA gene: C, chloroplast; M, mitochondrion; N, nucleus. Introns in archaea and bacteria are defined as
belonging to the nucleus. The only known rRNA intron in a bacterial genome occurs at position 1931 in the 23 S rRNA.
c
Position of nucleotide immediately prior to intron in Escherichia coli reference sequence. TBD, to be determined. These intron
sequences were published without flanking exon sequence and their insertion sequences could not be determined.
d
Number of intron sequences presently known at that position.
38 rRNA Introns in 3D
5. the total number of intron sequences. Conse-
quently, the general trends in the data are likely to
remain the same.
Non-random distribution of introns in
rRNA genes
Within 16 S (and 16 S-like) rRNA genes, 920
intron sequences are distributed over 84 sites,
among 1542 E. coli positions in the mature rRNA.
Among 23 S (and 23 S-like) rRNA genes, 325
introns are found at 69 sites, out of 2904 E. coli pos-
itions. Although 23 S rRNA is twice the length of
the 16 S rRNA, threefold more introns have been
documented in 16 S rRNA genes. This apparent
bias is largely due to unequal sampling of rRNA
sequences. As of February 2002, the CRW database
contains 7527 complete 16 S rRNA sequences and
960 complete 23 S rRNA sequences. When normal-
ized against the number of reported rRNA
sequences, the 16 S and 23 S rRNAs have approxi-
mately the same number of introns per rRNA
nucleotide, with ,1024
introns per sequence per
nucleotide in each gene.
Comparison of the data with a Poisson distri-
bution based on the mean frequency of rRNA
introns shows that many fewer sites contain
introns than would be expected if introns were
inserted randomly within rRNA genes (Figure 1).
A few sites in the rRNA account for a large fraction
of the intron sequences in the CRW database
(Table 1). These include positions 516, 943, 1506
and 1516 in the 16 S rRNA, with more than 100
known introns each, and positions 798, 2449, and
2500 in the 23 S rRNA, which each have more
than 25 intron sequences. This skewed distribution
persists even when sequence entries from closely
related species or strains are counted only once.
Hence, the presence of many intron sequences at a
few sites in the rRNA is not due to biased
sampling of rRNA sequences.
The tendency of introns to occur in particular
positions of the rRNA can be explained by two
mechanisms; (1) vertical inheritance of introns
from ancestral genes, or (2) preferential horizontal
transfer of intron sequences to certain target sites.
The presence of similar introns at the same
position in the rRNAs of related organisms is
usually attributed to stable inheritance of the
intron within the lineage. Examples of this inheri-
tance include 54 group IE introns that occur at
position 1199 of Ascomycota nuclear 16 S rRNAs35
and 128 group I introns at position 1516 in the
16 S rRNA genes of Lecanorineae (Table 1).
In contrast, different subclasses of introns at the
same rRNA position, or closely related introns
in unrelated organisms, are best explained by
horizontal transfer of the intron sequences. For
example, group IC1 introns with a distinctive
structural motif are inserted after position 516 in
the 16 S rRNA of red and brown algae but are not
found in intermediate relatives on the phylogenetic
tree.19
This suggests that the introns were acquired
independently after the red and brown algae
diverged. Introns have probably been inserted
(and lost) at position 516 many times during
evolution,14
as introns from two structural classes
(IC1 and IE) have been identified at this position
in four phylogenetic groups.5
In the 23 S
rRNA, nine group I introns corresponding to three
different subclasses were found at position 1931
in the mitochondrial, chloroplast and nuclear
rDNA of diverse organisms.16,17,36
Thus, the
phylogenetic data suggest that introns are repeat-
edly inserted at the same positions in rRNA
genes. Therefore, the prevalence of introns at
certain positions in rRNA genes is due not only
to inheritance of a few stable introns but also to
site-selective transposition and retention of introns.
Intron positions at the functional heart of
the ribosome
To determine whether the location of intron sites
depends on the higher-order structure of the
Figure 1. Distribution of introns in rRNAs. Histogram
of the number of positions in the (a) 16 S rRNA or (b)
23 S rRNA with a given number of intron insertions.
Positions are numbered according to the sequence of the
E. coli rRNAs. Shaded bars indicate the actual number
of intron-containing positions (Table 1); lines indicate
the expected number of intron-containing positions
expected from a Poisson distribution, based on the mean
frequency of introns in rRNA sequences. Insets show an
expansion of the y-axis.
rRNA Introns in 3D 39
6. rRNA, intron-containing positions were mapped
onto secondary structures of the 16 S and 23 S
rRNAs determined from comparative sequence
analysis (Figures 2(a) and 3(a)).3,5,37
The intron
positions were also visualized on three-dimen-
sional structures of the 30 S and 50 S ribosomal
subunits recently determined by X-ray crystal-
lography28,29
(Figures 2(b) and 3(b) and Further
information; see Methods.
Introns are found in every domain of the 16 S
rRNAs. A large number of intron-containing sites
lie near highly conserved regions that function in
decoding and translocation, such as the 530 loop,
the “switch helix” (nucleotides (nt) 885–912), and
the 30
-terminal helix (nt 1506–1529). Other introns
are found near residues that form the ribosome
subunit interface, such as four intron insertions
within the 790 loop (Figure 2(a)). When mapped
onto the structure of the Thermus thermophilus
30 S ribosome29
(Figure 2(b)), intron-containing
positions are concentrated towards the center of
the subunit, as suggested by their locations in the
secondary structure. A third of intron positions
lie on the face of the 30 S particle that contacts the
50 S subunit. Strikingly, positions with large
numbers of introns (516, 788, 943, 1199, 1506 and
Figure 2. Location of introns in
16 S rRNAs. (a) Secondary structure
of 16 S rRNA, showing nucleotides
that are present in more than 95%
of known sequences.5,37
Red dot,
$98% conserved; gray dot, 90–
98% conserved; open circle, ,90%
conserved. Positions for which an
intron is documented are numbered
according to the sequence of the
E. coli 16 S rRNA and colored
according to intron type: green,
group I; red, group II; cyan,
archaeal; blue, spliceosomal;
orange, other; black, multiple intron
types. Nucleotides that are within
10 A˚ of mRNA or tRNA bound
at the A, P or E sites42
are shaded
yellow. Brackets indicate positions
that are not conserved among all
rRNA sequences. The asterisk (p)
indicates an insertion relative to
the E. coli sequence. (b) Three-
dimensional structure of the 30 S
ribosomal subunit (1FJF),29
with
intron-containing positions colored
according to intron type as in (a).
Large spheres represent positions
with more than 100 known intron
sequences. The 16 S rRNA is
shown as light gray ribbon; proteins
as pink ribbons.
40 rRNA Introns in 3D
7. 1516) all lie near functionally important residues at
the subunit interface.
Among 23 S rRNA genes, introns are also found
near functionally important positions in domains
II, IV and V of the 23 S rRNA (Figure 3(a)).
In domain IV of the 23 S rRNA, many introns are
found in sequences that form the interface with
the 30 S subunit, with a large number of insertions
near helix 69 (nt 1905–1920) at the center of the
subunit (Figure 3). This stem–loop interacts with
tRNAs bound in the A and P sites, and makes
contacts with 16 S rRNA residues in the decoding
site of the ribosome.38,39
In three-dimensional
views of the Haloarcula marismortui 50 S ribosome,28
the intron-containing positions in domain IV can
be seen running across the central ridge and
down the face that contacts the 30 S subunit
(Figure 3(b)).
Many introns are inserted adjacent to highly
conserved nucleotides in domain V of the 23 S
rRNA that form the peptidyl transferase center of
the ribosome (Figure 3(b)). These nucleotides line
both sides of the peptidyl transferase cleft (Figure
3(b)), placing the sites of intron insertions in close
proximity to the acceptor stems of bound tRNAs.
Among nucleotides deep in the peptidyl transfer-
ase cleft are several positions that frequently
precede intron sites, such as nt 2449 and 2500
(Table 1).
A smaller number of introns are also found
throughout domain II of 23 S rRNA genes (Figure
3). Several introns are found in the L11-binding
region (nt 1050–1100), which interacts with
elongation factors in the 70 S ribosome.40,41
Surpris-
ingly, no introns were found at rRNA positions
that face the solvent side of the 50 S subunit or in
the bottom surface created by domains III and VI
of the 23 S rRNA. Thus, intron-containing positions
in the 23 S rRNA are concentrated in the center of
the 50 S particle and on the surface that contacts
the 30 S subunit.
Three-dimensional clusters of intron sites
To quantify the extent to which intron insertion
sites are clustered within the three-dimensional
structure of the 70 S ribosome, we computed a
center of mass from the coordinates of the 30
phos-
phate group of each intron insertion site. The
standard deviations of the x, y and z coordinates
were summed to create a “cluster score,” with a
lower number representing a tighter group (see
Methods). As illustrated in Figure 4(a), nucleotides
that precede introns are closer to each other than
the same number of randomly selected phosphate
groups in the rRNA. This is particularly true of
positions that contain large numbers of introns.
Hence, intron-containing positions are more tightly
clustered in space when each coordinate is
weighted by the number of rRNA genes containing
an intron at that position (Figure 4(a)).
Proximity to conserved rRNA nucleotides
To analyze the extent to which introns are
present at conserved rRNA sites, the conservation
of nucleotides 50
and 30
of each intron insertion
site was determined. The level of conservation
was extracted from alignments of rRNA sequences
representing organisms from the three primary
phylogenetic domains, plus chloroplasts and
mitochondria,5
and is available at the CRW Site
(see Methods). Nucleotides that are more than
98% identical (category A) or 90–98% identical
(category B) are highlighted in red and gray in the
secondary structure diagrams in Figures 2(a) and
3(a), and in space-filling representations of the
ribosomal subunits in Figure 5.
Only 12% and 5% of all 16 S and 23 S rRNA
nucleotides, respectively, are conserved in more
than 98% of all known rRNA sequences (category
A). In contrast, 21% of 16 S and 25% of 23 S rRNA
nucleotides that precede an intron-insertion site
are conserved at this level (Table 2(A)). We observe
a similar bias in the number of category B nucleo-
tides (90–98% identical) preceding intron positions
(Table 2(A)). Alternatively, we counted the number
of intron-containing positions that occur near a
category A nucleotide (Table 2(B)). About a third
(36%) of 16 S rRNA intron positions were adjacent
to a category A nucleotide, compared with 18% of
all 16 S rRNA nucleotides. In the 23 S rRNA, 32%
of intron-containing positions were adjacent to a
category A nucleotide, compared with 8% overall.
Positions with many introns (16 S positions 516,
788, 943, 1199, 1506, 1512, 1516; 23 S positions 798,
1921, 2449, 2500) are even more likely to be next to
highly conserved nucleotides than positions that
contain only one or a few known introns. This
bias is quite strong, particularly among 16 S rRNA
sequences. Thus, introns are much more likely to
lie within conserved regions of the rRNA than
expected if they were randomly inserted in rRNA
genes.
Although introns tend to occur in conserved
regions of the rRNA, not all highly conserved
regions contain known introns. The most con-
served regions of the 16 S and 23 S rRNAs that do
not contain any known introns as of February
2002 are the 250 region in domain I and the alpha-
sarcin/ricin loop in domain VI (2650–2670) of the
23 S rRNA, and the 1400 region of the 16 S rRNA
that forms part of the mRNA decoding center.
In fact, the lack of introns in domain VI of the
23 S rRNA is striking, given its conservation and
functional importance.
Proximity to tRNA binding sites
Most introns tend to be inserted near residues
that form tRNA binding sites (Figures 2 and 3).
This trend was evaluated by computing the frac-
tion of intron sites within a certain distance of the
A, P or E sites of the 70 S ribosome (see Methods).
As shown in Figure 6, intron-containing positions
rRNA Introns in 3D 41
9. are closer in space to bound tRNAs than average
positions in the 16 S or 23 S rRNAs. Among 153
positions that contain introns, 26 are within 10 A˚
of a tRNA site and include rRNA nucleotides that
make direct interactions with bound tRNAs.31
Roughly 60% of introns lie within 20 A˚ of the A, P
or E sites, and 90% are within 40 A˚ . In contrast,
the mean distance of all rRNA residues from the
tRNA sites is 50 A˚ . Nearly all of the rRNA sites
that contain a large number of introns are within
20 A˚ of a bound tRNA. These include positions
516, 943 and 1506 in the 16 S rRNA and 2449 and
2500 in the 23 S rRNA. Position 1506 in the 16 S
rRNA is even closer (10 A˚ ) to the mRNA channel.42
The proximity of intron-containing positions
to tRNA binding sites explains the clustering of
introns within the three-dimensional structure of
the 70 S ribosome and is congruent with their
proximity to highly conserved rRNA residues.
A corollary of the association between introns
and functional sites in the ribosome is that intron-
containing positions occur in RNA-rich areas of
the ribosome. Qualitative examination of the 30 S
ribosome structure showed that very few intron-
containing positions interact directly with riboso-
mal proteins. Almost no intron-containing
positions in the 23 S rRNA contact proteins, as
most proteins bind the solvent face of the large
subunit,28
which is devoid of introns.
Comparison of intron types
Introns belonging to different classes (group I, II,
archaeal and spliceosomal) are rarely inserted at
precisely the same position within the rRNA.5
Two exceptions are position 788 in the 16 S rRNA,
which contains group I and group II introns
(Figure 2(a) and Table 1A), and position 787 in the
Figure 4. Three-dimensional
clustering of rRNA intron sites
within the 70 S ribosome. Cluster
scores were computed as described
in Methods; a lower score indicates
a narrower distribution of x, y and
z coordinates. (a) Comparison of
rRNA positions in the 16 S and
23 S rRNAs (70 S), 23 S rRNA only
(50 S), or 16 S rRNA only (30 S).
Dark gray, all rRNA positions; light
gray, intron-containing positions;
white, intron-containing sequences;
hatched, P-site tRNA. (b) Compari-
son of intron types. Dark gray, 70 S
ribosome; light gray, 50 S ribosome;
white, 30 S subunit. All positions
corresponds to all rRNA residues;
reverse splicing refers to positions
that were experimentally found to
react with the Tetrahymena group I
intron.47,48
Figure 3. Location of introns in 23 S rRNAs. (a) Secondary structure of 23 S rRNA, showing nucleotides that are
present in more than 95% of known sequences.3,5
Nucleotides are represented as in Figure 2(a). Positions that contain
a documented intron are numbered according to the sequence of the E. coli 23 S rRNA and colored according to intron
type as in Figure 2(a). (b) Three-dimensional structure of the 50 S ribosome (1JJ2),76
colored as in Figure 2(b). Spheres
represent intron-containing positions; large spheres indicate positions with more than 29 known intron sequences.
rRNA Introns in 3D 43
10. 23 S rRNA, which contains group II and
spliceosomal introns (Figure 3(b) and Table 1B). A
few sites contain both group I and unclassified
introns (16 S rRNA positions 516 and 943; 23 S
positions 1923 and 1974). As each intron type has
a different splicing mechanism, distinct insertion
sites may reflect different constraints on splice-site
recognition. For example, the 50
splice site of
group I introns is nearly always preceded by a
U,43,44
whereas group II and archaeal exon bound-
aries are variable in sequence.26,45
Although their precise insertion sites do not
overlap, most introns are inserted near rRNA resi-
dues that form the center of the 70 S ribosome
(Figures 2(b) and 3(b)). Nucleotides near the center
of the ribosome tend to be conserved, and group I,
group II and archaeal introns are particularly likely
to be inserted at locations within three positions of
a category A nucleotide (Table 2B). These data
suggest that the evolutionary forces that shape the
distribution of group I, group II and archaeal
introns within rRNA genes originate from the
sequence and structure of the rRNA itself rather
than the type of intron.
In contrast, spliceosomal introns are less
clustered with respect to the three-dimensional
structure of the ribosome than group I and group
II introns (Figure 4(b)). Moreover, they are less
likely to be near a highly conserved nucleotide
than other types of introns (Table 2B), and more
likely to be found in residues far from tRNA
binding sites. For example, only one spliceosomal
intron is found in 23 S rRNA domain IV (1849),
although this region contains many group I and
group II intron insertions (Figure 3(a)). As spliceo-
somal introns do not have a conserved secondary
structure, their splicing may be less sensitive to
the structure of the rRNA exons than group I,
group II and archaeal introns, which must fold
into a precise structure in order to be spliced.
Solvent accessibility of intron positions
Introns may preferentially exist near rRNA
residues that interact with tRNAs, because these
residues are on the surface of the ribosome. If
introns are inserted into the rRNA by reverse
splicing, we would expect them to integrate more
frequently into accessible positions of the
rRNA.23,46
Alternatively, introns may be retained
more frequently at exposed positions where they
might not interfere with ribosome assembly.
To address this question, the solvent-accessible
surface area (ASA) of each atom was computed
from the coordinates for the T. thermophilus
30 S ribosome and the 50 S subunit from
H. marismortui, as described in Methods.
The 30
oxygen atom was used as a proxy for
intron insertion sites, although similar results
were obtained with other backbone atoms. As
expected, many 30
oxygen atoms are fully buried
(ASA ¼ 0 A˚ 2
), while others are at least partially
exposed to solvent (ASA . 4 A˚ 2
) (Figure 7). The
latter correspond to residues that are on the surface
Figure 5. Conservation of rRNA. Intron positions (yellow spheres) are indicated on a space-filling model of the ribo-
some. Red, rRNA nucleotides that are .98% conserved among all sequences (category A); pink, nucleotides that are
90–98% conserved (category B).5
Atoms are rendered at twice the van der Waal’s radius; intron positions are rendered
four times larger. (a) 30 S (front view). (b) 50 S (crown view). The figure was prepared with Insight II.
44 rRNA Introns in 3D
11. of the ribosomal subunit. Contrary to our expec-
tations, 30
oxygen atoms preceding intron-contain-
ing sites were exposed to solvent in the same
proportion at all rRNA positions (Figure 7). From
these data, we conclude that intron-containing
positions are not systematically biased towards
rRNA positions that are solvent-accessible in
mature subunits.
Visual inspection of the three-dimensional struc-
tures of the 30 S and 50 S subunits (Figures 2 and
3) and the data in Figure 7 show that introns are
inserted in both accessible and buried positions.
Some regions of the 23 S rRNA that contain many
introns are highly exposed to solvent in the 50 S
ribosome, such as 1080 and 1915. Others, such as
position 2449, have an average accessibility, while
a few, such as 2500, are buried deeply within the
50 S subunit. The 30 S subunit is quite flat, so that
even residues that are buried in the static structure
of the ribosome are not far from the surface of the
Table 2. Conservation of rRNA near intron sites
16 S 23 S
nt %Total nt %Total
A. Conservation of rRNA residues prior to intron insertion sitesa
All rRNA residues
.98% 178 11.5 150 5.2
90–98% 175 11.3 203 7.0
80–90% 116 7.5 168 5.8
Total 1542 100.0 2904 100.0
nt prior to:
All introns
.98% 18 21.4 17 24.7
90–98% 14 16.7 12 17.4
80–90% 12 14.3 5 7.2
Total 84 100.0 69 100.0
Group I
.98% 11 28.2 10 24.4
90–98% 4 10.3 4 9.8
80–90% 4 10.3 4 9.8
Total 39 100.0 41 100.0
Group II
.98% 1 33.3 3 37.5
90–98% 1 33.3 2 25.0
80–90% 0 0 0 0
Total 3 100.0 8 100.0
Archaeal
.98% 4 28.6 3 50.0
90–98% 7 50.0 2 33.3
80–90% 1 7.1 0 0
Total 14 100.0 6 100.0
Spliceosomal
.98% 0 0 1 7.1
90–98% 2 9.1 4 28.6
80–90% 7 31.8 1 7.1
Total 22 100.0 14 100.0
B. Number of intron-containing positions adjacent to highly conserved
(.98%) rRNA residues
Distance to nearest conserved rRNA position:
All rRNA residues
Adjacent to at least one 274 17.8 240 8.3
Within 2 nt 351 22.8 308 10.6
Within 3 nt 417 27.1 359 12.4
All introns
Adjacent to at least one 30 35.7 22 31.9
Within two nt 41 48.8 33 47.8
Within three nt 50 59.5 40 57.9
Flanked by two 6 7.1 9 13.0
Group I
Adjacent to at least one 19 48.7 12 29.3
Within 2 nt 24 61.5 19 46.4
Within 3 nt 29 74.4 22 53.7
Flanked by two 4 10.3 4 9.8
Group II
Adjacent to at least one 1 33.3 4 50.0
Within 2 nt 3 100.0 6 75.0
Within 3 nt 3 100.0 7 87.5
Flanked by two 0 0 2 25.0
Archaeal
Adjacent to at least one 7 50.0 4 66.7
Within 2 nt 8 57.1 4 66.7
Within 3 nt 8 57.1 4 66.7
Flanked by two 1 7.1 3 50.0
(continued)
Table 2 Continued
16 S 23 S
nt %Total nt %Total
Spliceosomal
Adjacent to at least one 1 4.5 2 14.3
Within 2 nt 4 18.2 4 28.6
Within 3 nt 7 31.8 7 50.0
Flanked by two 0 0 0 0
a
Conservation levels were calculated based on a large
sample of archaeal, bacterial, and eukaryotic nuclear, mito-
chondrial and chloroplast rRNA sequences.5
The three conserva-
tion ranges (.98%, 90–98% and 80–90%) were determined for
positions that are present in more than 95% of all sequences.
Figure 6. Proxmity of intron sites to tRNA bound in
the 70 S ribosome. The total number of rRNA positions
within a given cutoff distance of tRNA bound in the A,
P or E site of the 70 S ribosome was calculated as
described in Methods. Intron-containing positions were
weighted by the number of rRNA sequences with an
intron at that position. Continuous line, all 16 S rRNA
positions; long dash, 16 S introns; medium dash, 23 S
introns; short dash, all 23 S rRNA positions.
rRNA Introns in 3D 45
12. particle. All the same, introns are found in 16 S
rRNA positions that are among the least accessible
in the 30 S crystal structure.
The similarity between the ASA values for all
rRNA positions and those containing introns
remained constant when spheres of radius 1.4 A˚
and 5 A˚ were used to compute the ASA. We also
observed no difference between the accessibilities
of intron insertion sites and average rRNA nucleo-
tides in the absence of ribosomal proteins,
although the mean accessibility of all residues
increased. Taken together, these data suggest that
intron insertion sites are not exclusively found in
residues that are highly exposed in the static
structures of the 30 S and 50 S ribosomes. It is pos-
sible, however, that many intron-containing
positions are exposed by conformational changes
during translation or during assembly of ribosomal
particles.
Reverse splicing target sites
Previous work showed that the Tetrahymena
group I intron can integrate partially or fully into
many sites in the 23 S rRNA by reverse splicing
when overexpressed in E. coli cells.47,48
An interest-
ing question is whether experimentally observed
reverse splicing targets are clustered in the same
rRNA regions as naturally occurring introns. In
contrast to the sites of natural introns, sites that
react with the Tetrahymena intron are not only
found on the subunit interface but also lie on the
“back” face of the 50 S ribosome, away from the
30 S subunit (Figure 8). The reaction sites are less
tightly clustered with each other than the positions
of natural introns (Figure 4) and not localized
around the tRNA binding sites. They were also
slightly less solvent-accessible than average,
suggesting that accessibility is not a prerequisite
for insertion of introns by reverse splicing. The
difference in the spatial distribution of naturally
occurring introns and reverse splicing targets
suggests that other factors determine the positions
of rRNA introns, such as selective retention after
intron insertion or protein-dependent transposition
mechanisms.
Discussion
The extensive rRNA intron sequence collection
available at the Comparative RNA Web (CRW)
Site5
is the foundation of our investigation of the
distribution of introns within the 16 S and 23 S
rRNAs. Positions of the rRNA that are presently
known to contain introns were mapped to the
secondary structures of the rRNA determined
from comparative sequence analysis3,5,37
and
the three-dimensional structures of the 30 S, 50 S
and 70 S ribosomes determined by X-ray
crystallography.28 – 31
Our results, which are based on the analysis of
more than 1250 rRNA intron sequences, reveal
that introns are not inserted randomly within the
16 S and 23 S rRNA genes. Instead, they are
clustered near tRNA binding sites, which span the
interface between the small and large subunits of
the 70 S ribosome. Surprisingly, intron-containing
residues are not more solvent-accessible in the
30 S and 50 S subunits than typical rRNA residues.
Thus, while the distribution of introns is coupled
to the sequence and structure of the rRNA, it does
not directly correlate with the accessibility of
the splice junction in static three-dimensional
structures of the ribosome.
Horizontal transfer of intron sequences
Comparative analyses of intron sequences
within specific phylogenetic groups have con-
cluded that introns at the same position of the
rRNA are usually more closely related to one
another than introns at different positions.13,14,35,49–51
Figure 7. Surface accessibility of intron positions in
mature ribosomal subunits. The ASA of 30
oxygen atoms
in the 50 S and 30 S ribosomal subunits was calculated
as described in Methods. Histograms show the percen-
tage of rRNA positions with a given ASA value. Shaded
bars, intron-containing positions; white bars, all rRNA
positions. (a) 16 S rRNA in the 30 S subunit. (b) 23 S
rRNA in the 50 S subunit.
46 rRNA Introns in 3D
13. This has led to the view that some introns are
retained for long periods of evolution after their
initial insertion into a specific position in the rRNA
gene. On the other hand, introns are periodically
lost from certain lineages.13,14
More rarely, they are
regained or transferred to a new lineage.15 – 19,36
Hence, intron mobility may be required to ensure
survival and the spread of intron sequences to
new organisms.10
Besides ensuring intron retention, frequent
deletion and reinsertion of intron sequences may
result in different phylogenetic trees for rRNA
exons and introns, as observed for the group IC1
intron in the rDNA of Tetrahymena.52
This type of
dynamic flux has been invoked to explain
sequence heterogeneity of non-LTR retrotranspo-
sons in insect rDNA.53
As most insertion events
recur at the same site in the rRNA with only
occasional transposition to a new site,54
the
position of the retroelement in the rRNA is main-
tained, although its sequence may vary. If introns
are continually deleted and reinserted, their final
distribution will depend heavily on the selectivity
of retention and the target-specificity of the inser-
tion mechanism. These factors are discussed below.
Target-site selection during intron transposition
Many group I and group II introns and some
archaeal introns have acquired open reading
frames that encode homing DNA endonucleases.10
Although these endonucleases usually recognize
the same position of an intron-less allele of the
same gene, cross-reaction with related sequences
could enable transfer of the intron to homologous
positions in the rRNA genes of a different organ-
ism or occasional transposition to ectopic sites.55 – 57
Because of the large recognition sites of most
homing endonucleases,58
this process would be
more effective in conserved regions of the rRNA,
contributing to the prevalence of introns at those
sites.
The feasibility of intron transfer was demon-
strated by integration of group I introns from
Physarum polycephalum and Tetrahymena thermophila
into the rDNA of yeast upon co-expression of
the I-PpoI endonuclease.56
Because the rRNA
sequences flanking the intron are nearly identical
in these species, the introns are spliced correctly in
yeast.59
Similarly, reverse transcriptases encoded
by group II introns normally promote movement
of the intron to an allelic site, but in some circum-
stances can transpose the intron to an ectopic
site.60 – 63
Although the substrate specificity of proteins
associated with intron mobility might account for
the prevalence of introns in conserved regions of
the rRNA, this mechanism of intron transposition
is inconsistent with the sequence data. First, the
majority of rRNA introns do not contain open
reading frames that encode endonucleases.5
At
some sites that contain many introns, such as 1506
in the 16 S rRNA, only a few of the extant introns
have open reading frames (ORFs) greater than
500 nt.5
While all introns may have contained
ORFs at one time that were subsequently deleted
by non-homologous recombination, only a few
cases of vestigial ORFs have been documented in
rRNA introns (in Naegleria64
and in the order
Bangiales14
). Second, this mechanism requires that
Figure 8. Locations of reverse splicing targets in E. coli 23 S rRNA. Integration sites of the Tetrahymena group I
intron RNA when overexpressed in E. coli47,48
are indicated by white spheres on the crown (left) and back (right)
views of the 50 S subunit.28
Large spheres indicate frequent reaction sites. The 23 S rRNA is shown as a ribbon colored
by domain: blue, domain I; cyan, domain II; yellow, domain III; green, domain IV; red, domain V; pink, domain VI;
magenta, 5 S rRNA. Proteins are not shown.
rRNA Introns in 3D 47
14. the endonuclease be properly expressed in the
recipient organism, which could be problematic in
cases of intron transfer between organelles.16
Transposition via reverse splicing
An alternative to protein-dependent mecha-
nisms is that introns integrate into rRNAs by
reverse splicing.46
This could lead to transposition
of the intron to novel sites in the genome, follow-
ing reverse transcription and recombination with
the intron-containing cDNA. An appealing aspect
of this mechanism is that integration of the intron
into the rRNA would be immediately sensitive to
the conformation of the rRNA.24
Another advan-
tage is that group I reverse splicing is much less
site-specific than reactions catalyzed by homing
endonucleases.48
Despite the appeal of RNA-based intron trans-
position, the biochemical support for such a
mechanism is weak. Reverse splicing of the
Tetrahymena group I intron into rRNA in E. coli
was observed by RT-PCR,65
but stable integration
of a group I intron by reverse splicing has not yet
been demonstrated. Group II introns also reverse
splice, but the target is allelic DNA rather than
RNA.66
In addition, we find no correlation between
the frequency of introns and solvent accessibility of
the rRNA insertion site. Thus, if reverse splicing
contributes significantly to the spectrum of introns
found in rRNA genes, then the intron must react
with pre-rRNA that is not fully assembled into a
ribosomal complex or the target rRNA must transi-
ently unfold. Finally, reverse splicing products of
the Tetrahymena intron in the E. coli 23 S rRNA
are distributed differently than natural rRNA
introns with respect to the structure of the 50 S sub-
unit. This suggests that the ability of certain
sequences to serve as reverse splicing substrates is
not sufficient to describe the actual distribution of
rRNA introns.
Retention of introns in dynamic regions of
the rRNA
An alternative possibility is that introns are
inserted randomly within the rRNA genes, but
retained only at certain positions. Many random
integration products are expected to be unstable,
either because of recombination with cDNA or
other rDNA repeats or simply because the intron
is not spliced effectively from its new location,
inactivating the rRNA. When a homing endo-
nuclease is continually expressed, aggressive
homing will permit even deleterious rRNA inser-
tions to be maintained.4
However, once the ORF is
not expressed (as in most rRNA introns), introns
that reduce fitness will be lost. Accurate splicing,
degradation of the excised intron RNA (to prevent
cleavage of the spliced exons)67
and proper folding
of the rRNA are all necessary to maintain host cell
functions.
It is interesting to consider whether introns are
more easily retained in positions near the subunit
interface and tRNA binding sites because these
nucleotides undergo structural changes during
translation.68
Flexible sites should accommodate
introns more easily than rigid sites, because in
many cases the secondary structure of the mature
rRNA must unfold to permit splice site
recognition.24,26
Conversely, the presence of highly
structured introns may either interfere less
severely with ribosome assembly or even enhance
folding of the rRNA.
A striking example of introns in a structurally
dynamic region of the rRNA occurs in domain IV
of the 23 S rRNA. Helix 69 (nt 1905–1920) is dis-
ordered in crystals of the 50 S ribosome, suggesting
that it remains mobile under these conditions.28,30
In the 70 S ribosome, this stem–loop and adjacent
sequences form an important bridge to the decod-
ing site in the 30 S subunit.31
Many group I introns
and a few archaeal introns are found between
positions 1915 and 1932 of the 23 S rRNA (Figure
3(a)). Experiments on the group I intron of
Tetrahymena (position 1925) have shown that the
conformation of the rRNA exons is intimately
linked to self-splicing activity.25,69
Similarly, refold-
ing of helix 69 is required for excision of the
archaeal intron at position 1927 in Staphylothermus
marinus.26
In addition to these examples, many other
intron-containing sites are in mobile regions of the
rRNA. For example, the L11-binding region of
the 23 S rRNA is disordered in crystals of 50 S
ribosomes,28,30
and is also highly mobile.31
Within
this region (nt 1050–1108), five sites contain group
I introns, and one site contains an archaeal intron.
Positions 2449 and 2500 in the 23 S rRNA are part
of the peptidyl transferase center and are thought
to undergo conformational changes during ribo-
some translocation.70
In the 16 S rRNA, many
introns are inserted in the 790 loop and in the 30
terminal helix (positions 1506, 1512 and 1516),
which forms an inter-subunit bridge near the
P-site tRNA.31
Position 516 contains a large number
of group I introns; this residue is part of the con-
served 530 loop that undergoes conformational
changes during mRNA decoding.71,72
Regulatory role for introns?
Finally, pre-rRNA splicing may even be an
additional mechanism for regulating ribosome
activity, providing another link between RNA
processing and translation. As introns are not
universally present among all members of a phylo-
genetic group, their function cannot be essential to
translation. Yet, they may provide useful regu-
latory functions under the growth conditions
encountered by specific organisms. Regulation
could occur during or after subunit assembly.
First, the presence of an intron in the pre-ribosomal
RNA could arrest ribosome assembly, either by
sterically blocking tertiary contacts (e.g. in the
48 rRNA Introns in 3D
15. interior of the 50 S subunit), or by altering local
secondary structures.24
As introns are rarely found
in ribosomal protein binding sites, unspliced
introns would affect protein binding only
indirectly via structural changes in the rRNA.
Second, introns could be required for modification
of the rRNA in some organisms, as in some
tRNAs.73,74
Third, introns could inhibit the trans-
lational activity of nascent subunits. By analogy to
proenzymes, splicing would be required for acti-
vation. Introns inserted in residues at the inter-
subunit interface, for example, might not prevent
assembly of individual subunits, but would
prevent the subunit from forming productive 70 S
ribosomes.75
In summary, our analysis confirms the statistical
correlation between the positions of rRNA introns
and the structure and conservation of the 16 S and
23 S rRNAs. It is now apparent that this bias results
in the preferential location of introns in conserved
rRNA residues that are clustered around tRNA
binding sites in the three-dimensional structure of
the 70 S ribosome. At present, it is difficult to
rationalize the rRNA intron distribution based
only on the selectivity of known intron insertion
mechanisms. This suggests that the extant
positions are selected by either a need to maintain
efficient splicing or the stringent requirements of
pre-rRNA maturation and ribosome assembly.
Both of these effects may favor introns in dynamic
regions of the rRNA.
Methods
rRNA intron database
The rRNA intron sequences were collected, analyzed,
and assembled into the relational database for the CRW
project, as described in detail.5
Only sequences that are
more than 90% complete and publicly available are
included in the database. As of February 2002, the
database contained 8865 rRNA sequences and 1285
annotated rRNA intron sequences. The positions of
rRNA introns are numbered according to the nucleotide
50
of the intron in the sequence of the E. coli 16 S or 23 S
rRNA (GenBank accession number J01695). Our determi-
nation of the intron types utilized the descriptions of
sequence and secondary structure of group I,6
group
IE,33
group II,45
spliceosomal34
and archaeal introns.26
The spliceosomal introns noted here are defined by
homology to pre-mRNA introns;9
the biochemical
mechanism of splicing has not yet been established.
Intron-containing rRNA positions in three-
dimensional ribosome structures
Structural data were obtained from the Protein Data
Bank (PDB).† Introns in 16 S or 16 S-like rRNA genes
were mapped onto the three-dimensional structure of
the T. thermophilus 30 S ribosome (1FJF).29
Introns in 23 S
or 23 S-like rRNA genes were mapped on the structure
of the H. marismortui 50 S ribosome (1JJ2).76
Three intron
positions (1956, 1958 and 1962) lie in regions of the 23 S
rRNA that are not visible in the electron density map.76
These were denoted with spheres at nearby residues:
1956 shown as 1950, 1958 as 1951, 1962 as 1965. Intron
positions were represented by a CPK rendering of the
30
-hydroxyl oxygen atom of the rRNA residue preceding
the intron insertion site. Molecular graphics analyses
were carried out using Insight II (Molecular Simulations
Inc.). Figures were prepared using Ribbons77
and Insight II.
Spatial distribution of rRNA positions
The spatial distribution of rRNA residues in the
T. thermophilus 70 S ribosome was determined from the
atomic coordinates of phosphorus atoms (1GIY and
1GIX).31
For each group of rRNA residues analyzed, we
computed the center of mass and the standard deviation
of the x, y and z coordinates. The “cluster score” is the
sum of the x, y and z standard deviations of the x, y and
z coordinates. A lower cluster score represents a tighter
three-dimensional clustering of nucleotides. Intron inser-
tions were represented by the 50
phosphate group of the
nucleotide 30
of the intron. To compute clustering of
intron sequences, all sequence entries in the CRW rRNA
intron database were used. To compute the clustering of
intron and rRNA positions, each rRNA position was
counted once, regardless of the number of intron
sequences that correspond to an insertion at that
position. Values for the 30 S and 50 S subunits were
computed using only residues from the 16 S or 23 S
RNAs, respectively.
Distance from tRNA binding sites
Subsets of rRNA positions were defined based on
their distance from the combined A, P and E site tRNAs
in the T. thermophilus 70 S ribosome (1GIY and 1GIX).
Distances were computed from the atomic coordinates
of the phosphorus atoms of the rRNAs and the bound
tRNAs,31
or the bound tRNAs and mRNA (see
website).42
† Distance cut-offs were set between 5 and
100 A˚ ; all rRNA phosphate groups are within 120 A˚ of
the tRNAs. The total number of rRNA residues and the
number of intron-containing sequences within the subset
defined by each distance cutoff are plotted in Figure 6.
These rRNA positions are listed on the website associ-
ated with this work (see below).
Solvent-accessibility
The ASA was computed for each atom in the 50 S
(1JJ2) and 30 S (1FJF) ribosomes, using the program
Calc-surf.78
A sphere of radius 1.40 A˚ was chosen to
simulate solvation by a water molecule. The surface
areas shown in Figure 7 were computed for each entire
subunit with protein side-chains, but without water
molecules and heteroatoms. The ASA values for 30
oxygen atoms were binned and plotted in Figure 7.
Further information available on website
Copies of the manuscript Figures and Tables and
additional Figures and Tables related to this work are
available from the CRW Site‡. The intron sites on the
† http://www.rcsb.org/pdb/
‡ http://www.rna.icmb.utexas.edu/ANALYSIS/
INT3D/
rRNA Introns in 3D 49
16. 16 S and 23 S rRNAs (as of February 2002) and related
information are shown on three sets of secondary
structure diagrams (PostScript and PDF formats) and
images of the three-dimensional crystal structures of the
ribosome.
Five dimensions of information are highlighted in this
presentation: (A) intron types (i.e. group I, group II,
archaeal, spliceosomal, and unknown); (B) the number
of introns per site (for 3D images only); (C) rRNA sites
within 10.0 A˚ of tRNA/mRNA; (D) extent of conserva-
tion of the 16 S and 23 S rRNAs; and (E) structural
domains from the 16 S and 23 S rRNA secondary
structure models are distinguished. The three-dimen-
sional images can be viewed interactively using
RasMol.79
Data tables include the source data for Tables
1 and 2, as well as additional data not discussed here.
Acknowledgements
This work was supported by grants from the
NIH (GM48207 to R.R.G. and GM46866 to S.A.W.),
NSF (MCB-0110252 to R.R.G.), the Welch Foun-
dation (F-1427 to R.R.G.) and The Institute for
Cellular and Molecular Biology at The University
of Texas at Austin (to R.R.G.).
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Edited by J. Doudna
(Received 30 April 2002; received in revised form 15 August 2002; accepted 19 August 2002)
52 rRNA Introns in 3D
