Background
In the 1830's, Charles Darwin's investigation of the
Galapagos finches led to an appreciation of the structural
characteristics that varied and were conserved among the
birds in this landmark comparative study. His analysis of
the finches' structural features was the foundation for his
theory on the origin and evolution of biological species [
1 ] . Today, 150 years later, our understanding of cells
from a molecular perspective, in parallel with the
technological advances in nucleic acid sequencing and
computer hardware and software, affords us the opportunity
to determine and study the sequences for many genes from a
comparative perspective, followed by the computational
analysis, cataloging, and presentation of the resulting
data on the World Wide Web.
In the 1970's, Woese and Fox revisited Darwinian
evolution from a molecular sequence and structure
perspective. Their two primary objectives were to determine
phylogenetic relationships for all organisms, including
those that can only be observed with a microscope, using a
single molecular chronometer, the ribosomal RNA (rRNA), and
to predict the correct structure for an RNA molecule, given
that the number of possible structure models can be larger
than the number of elemental particles in the universe. For
the first objective, they rationalized that the origin of
species and the related issue of the phylogenetic
relationships for all organisms are encoded in the
organism's rRNA, a molecule that encompasses two-thirds of
the mass of the bacterial ribosome (ribosomal proteins
comprise the other one-third). One of their first and most
significant findings was the discovery of the third kingdom
of life, the Archaebacteria (later renamed Archaea) [ 2 3 4
] . Subsequently, the analysis of ribosomal RNA produced
the first phylogenetic tree, based on the analysis of a
single molecule, that included prokaryotes, protozoa,
fungi, plants, and animals [ 4 ] . These accomplishments
were the foundation for the subsequent revolution in
rRNA-based phylogenetic analysis, which has resulted in the
sequencing of more than 10,000 16S and 16S-like rRNA and
1,000 23S and 23S-like rRNA genes, from laboratories trying
to resolve the phylogenetic relationships for organisms
that occupy different sections of the big phylogenetic
tree.
The prediction of tRNA structure with a comparative
perspective in the 1960's [ 5 6 7 8 9 ] and subsequent
validation with tRNA crystal structures [ 10 11 ]
established the foundation for Woese and Fox in the 1970's
to begin predicting 5S rRNA structure from the analysis of
multiple sequences. They realized that all sequences within
the same functional RNA class (in this case, 5S rRNA) will
form the same secondary and tertiary structure. Thus, for
all of the possible RNA secondary and tertiary structures
for any one RNA sequence, such as for
Escherichia coli 5S rRNA, the correct
structure for this sequence will be similar to the correct
secondary structure for every other 5S rRNA sequence [ 12
13 ] .
While the first complete 16S rRNA sequence was
determined for
E. coli in 1978 [ 14 ] , the first
covariation-based structure models were not predicted until
more 16S rRNA sequences were determined [ 15 16 17 ] . The
first 23S rRNA sequence was determined for
E. coli in 1980 [ 18 ] ; the first
covariation-based structure models were predicted the
following year, once a few more complete 23S rRNA sequences
were determined [ 19 20 21 ] . Both of these comparative
structure models were improved as the number of sequences
with different patterns of variation increased and the
covariation algorithms were able to resolve different types
and extents of covariation (see below). Initially, the
alignments of 16S and 23S rRNA sequences were analyzed for
the occurrence of G:C, A:U, or G:U base pairs that occur
within potential helices in the 16S [ 15 22 ] and 23S [ 19
] rRNAs. The 16S and 23S rRNA covariation-based structure
models have undergone numerous revisions [ 23 24 25 26 27
28 ] . Today, with a significantly larger number of
sequences and more advanced covariation algorithms, we
search for all positional covariations, regardless of the
types of pairings and the proximity of those pairings with
other paired and unpaired nucleotides. The net result is a
highly refined secondary and tertiary covariation-based
structure model for 16S and 23S rRNA. While the majority of
these structure models contain standard G:C, A:U, and G:U
base-pairings arranged into regular secondary structure
helices, there were many novel base-pairing exchanges (
e.g., U:U <-> C:C; A:A
<-> G:G; G:U <-> A:C;
etc. ) and base pairs that form
tertiary or tertiary-like structural elements. Thus, the
comparative analysis of the rRNA sequences and structures
has resulted in the prediction of structure and the
identification of structural motifs [ 29 ] .
Beyond the comparative structure analysis of the three
ribosomal RNAs and transfer RNA, several other RNAs have
been studied with this perspective. These include the group
I [ 30 31 32 33 ] and II [ 34 35 ] introns, RNase P [ 36 37
38 ] , telomerase RNA [ 39 40 ] , tmRNA [ 41 ] , U RNA [ 42
] , and the SRP RNA [ 43 ] . The comparative sequence
analysis paradigm has been successful in determining
structure over this wide range of RNA molecules.
Very recently, the authenticities of the ribosomal RNA
comparative structure models have been determined [Gutell
et al., manuscript in preparation]:
97-98% of the secondary and tertiary structure base pairs
predicted with covariation analysis are present in the
crystal structures for the 30S [ 44 ] and 50S [ 45 ]
ribosomal subunits. Thus, the underlying premise for
comparative analysis and our implementation of this method,
including the algorithms, the sequence alignments, and the
large collection of comparative structure models with
different structural variations for each of the different
RNA molecules (
e.g., 16S and 23S rRNAs) have been
validated.
The highly refined and accurate analysis of phylogenetic
relationships and RNA structure with comparative analysis
can require very large, phylogenetically and structurally
diverse data sets that contain raw and analyzed data that
is organized for further analysis and interpretation. With
these requirements for our own analysis, and the utility of
this comparative information for the greater scientific
community, we have been assembling, organizing, analyzing,
and disseminating this comparative information. Initially,
a limited amount of sequence and comparative structure
information was available online for our 16S (and 16S-like)
[ 46 47 ] and 23S (and 23S-like) ribosomal RNAs [ 48 49 50
51 52 ] and the group I introns [ 33 ] . In parallel, two
other groups have been providing various forms of ribosomal
RNA sequence and structure data (the RDP/RDP II [ 53 54 ]
and Belgium (5S/5.8S [ 55 ] , small subunit [ 56 57 ] and
large subunit [ 58 59 ] ) groups). With significant
increases in the amount of sequences available for the RNAs
under study here, improved programs for the analysis of
this data, and better web presentation software, we have
established a new "Comparative RNA Web" (CRW) Site
http://www.rna.icmb.utexas.edu/. This resource has been
available to the public since January 2000.
Results and Discussion
1. Comparative structure models
1A. Current structure models for reference
organisms
The first major category, Comparative Structure
Models http://www.rna.icmb.utexas.edu/CSI/2STR/contains
our most recent 16S and 23S rRNA covariation-based
structure models, which were adapted from the original
Noller & Woese models (16S [ 15 22 ] and 23S [ 19 ]
rRNA), and the structure models for 5S rRNA [ 12 ] ,
tRNA [ 5 6 7 8 9 ] , and the group I [ 32 ] and group
II [ 34 ] introns, as determined by others. This
collection of RNA structure models was predicted with
covariation analysis, as described at the CRW Site
Methods Section
http://www.rna.icmb.utexas.edu/METHODS/and in several
publications (see below).
Briefly, covariation analysis, a specific
application of comparative analysis (as mentioned
earlier), searches for helices and base pairs that are
conserved in different sequences that form the same
functionally equivalent molecule (
e.g., tRNA sequences). It was
determined very early in this methodology that the
correct helix is the one that contains positions within
a potential helix that vary in composition while
maintaining G:C, A:U, and G:U base pairs. As more
sequences for a given molecule were determined, we
developed newer algorithms that searched for positions
in an alignment of homologous sequences that had
similar patterns of variation. This latter
implementation of the covariation analysis helped us
refine the secondary and tertiary structure models by
eliminating previously proposed base pairs that are not
underscored with positional covariation and identifying
new secondary and tertiary structure base pairs that do
have positional covariation [ 19 70 71 72 ] . Our
newest covariation analysis methods associate
color-coded confidence ratings with each proposed base
pair (see reference structure diagrams and Section 2A,
"Nucleotide Frequency Tabular Display," for more
details). One exception to this is the tRNA analysis,
which was initially performed with the Mixy
chi-square-based algorithm [ 71 ] , and thus the color
codes are based on that analysis.
When implemented properly, covariation analysis can
predict RNA structure with extreme accuracy. All of the
secondary structure base pairs and a few of the
tertiary structure base pairs predicted with
covariation analysis [ 5 6 7 8 9 71 72 73 74 ] are
present in the tRNA crystal structure [ 10 11 ] . The
analysis of fragments of 5S rRNA [ 75 ] and the group I
intron [ 76 ] resulted in similar levels of success.
Most recently, the high-resolution crystal structures
for the 30S [ 44 ] and 50S [ 45 ] ribosomal subunits
have given us the opportunity to evaluate our rRNA
structure models. Approximately 97-98% of the 16S and
23S rRNA base pairs predicted with covariation analysis
are in these crystal structures (Gutell
et al., manuscript in
preparation). This congruency between the comparative
model and the crystal structure validates the
comparative approach, the covariation algorithms, the
accuracy of the juxtapositions of sequences in the
alignments, and the accuracy of all of the comparative
structure models presented herein and available at the
CRW Site. However, while nearly all of the base pairs
predicted with comparative analysis are present in the
crystal structure solution, some interactions in the
crystal structure, which are mostly tertiary
interactions, do not have similar patterns of variation
at the positions that interact (Gutell
et al., manuscript in
preparation). Thus, covariation analysis is unable to
predict many of the tertiary base pairings in the
crystal structure, although it does identify nearly all
of the secondary structure base pairings.
Beyond the base pairs predicted with covariation
analysis, comparative analysis has been used to predict
some structural motifs that are conserved in structure
although they do not necessarily have similar patterns
of variation at the two paired positions. Our analyses
of these motifs are available in the "Structure,
Motifs, and Folding" section of our CRW Site.
While the secondary structure models for the 16S,
23S and 5S rRNAs, group I and II introns, and tRNA are
available at the "Current Structure Models for
Reference Organisms" page, our primary focus has been
on the 16S and 23S rRNAs. Thus, some of our subsequent
analysis and interpretation will emphasize only these
two RNAs.
Each RNA structure model presented here is based
upon a single reference sequence, chosen as the most
representative for that molecule (Table 1); for
example,
E. coli is the preferred choice
as the reference sequence for rRNA (5S, 16S, and 23S),
based on the early and continued research on the
structure and functions of the ribosome [ 77 78 ] .
Each of the six structure models (5S, 16S and 23S rRNA,
group I and II introns, and tRNA) in the "Current
Structure Models for Reference Organisms" page
http://www.rna.icmb.utexas.edu/CSI/2STR/contains six or
seven different diagrams for that molecule: Nucleotide,
Tentative, Helix Numbering, Schematic, Histogram,
Circular, and Matrix of All Possible Helices.
Nucleotide: The standard format for
the secondary structure diagrams with nucleotides
(Figures 2A, 2B, and 2C) reveals our confidence for
each base pair, as predicted by covariation analysis.
Base pairs with a red identifier ("-" for G:C and A:U
base pairs, small closed circles for G:U, large open
circles for A:G, and large closed circles for any other
base pair) have the greatest amount of covariation;
thus, we have the most confidence in these predicted
base pairs. Base pairs with a green, black, grey, or
blue identifier have progressively lower covariation
scores and are predicted due to the high percentages of
A:U + G:C and/or G:U at these positions. The most
current covariation-based
E. coli 16S and 23S rRNA
secondary structure models are shown in Figures 2A, 2B,
and 2C. Note that the majority of the base pairs in the
16S and 23S rRNA have a red base pair symbol, our
highest rating. These diagrams are the culmination of
twenty years of comparative analysis. Approximately
8500 16S and 16S-like rRNA sequences and 1050 23S and
23S-like rRNA sequences were collected from all
branches of the phylogenetic tree, as shown in Section
2, "Nucleotide Frequency and Conservation Information"
and in Table 2. These sequences have been aligned and
analyzed with several covariation algorithms, as
described in more detail in the "Predicting RNA
Structure with Comparative Methods" section of the CRW
Site http://www.rna.icmb.utexas.edu/METHODS/and in
Section 2A. All of the secondary structure diagrams
from the "Current Structure Models for Reference
Organisms" page are available in three formats. The
first two are standard printing formats, PostScript
http://www.adobe.com/products/postscript/main.htmland
PDF
http://www.adobe.com/products/acrobat/adobepdf.html.
The third, named "bpseq," is a simple text format that
contains the sequence, one nucleotide per line, its
position number, and the position number of the pairing
partner (or 0 if that nucleotide is unpaired in the
covariation-based structure model).
Tentative: In addition to the 16S
and 23S rRNA structure models, we have also identified
some base pairs in the 16S and 23S rRNAs that have a
lower, although significant, extent of covariation.
These are considered 'tentative' and are shown on
separate 16S and 23S rRNA secondary structure diagrams
http://www.rna.icmb.utexas.edu/CSI/2STR/. These base
pairs and base triples have fewer coordinated changes
(or positional covariations) and/or a higher number of
sequences that do not have the same pattern of
variation present at the other paired position.
Consequently, we have less confidence in these putative
interactions, in contrast with the interactions
predicted in our main structure models.
The
Helix Numbering secondary structure
diagrams illustrate our system for uniquely and
unambiguously numbering each helix in a RNA molecule.
Based upon the numbering of the reference sequence,
each helix is named for the position number at the 5'
end of the 5' half of the helix. For example, the first
16S rRNA helix, which spans
E. coli positions 9-13/21-25, is
named "9;" the helix at positions 939-943/1340-1344 is
named "939." This numbering system is used in the
Nucleotide Frequency Tabular Display tables (see
below). The
Schematic versions of the reference
structure diagrams replace the nucleotides with a line
traversing the RNA backbone.
The
"Histogram" and
"Circular" diagram formats
http://www.rna.icmb.utexas.edu/CSI/2STR/both abstract
the global arrangement of the base pairs. For the
histogram version (Figure 2D), the sequence is
displayed as a line from left (5') to right (3'), with
the secondary structure base pairs shown in blue above
the sequence line; below this line, tertiary structure
base pairs and base triples are shown in red and green,
respectively. The distance from the baseline to the
interaction line is proportional to the distance
between the two interacting positions within the RNA
sequence. In contrast, in the circular diagram, the
sequence is drawn clockwise (5' to 3') in a circle,
starting at the top. Secondary and tertiary base-base
interactions are shown with lines traversing the
circle, using the same coloring scheme as in the
histogram diagram. The global arrangement and
higher-order organization of the base pairs predicted
with covariation analysis are revealed in part in these
two alternative formats. The majority of the base pairs
are clustered into regular secondary structure helices,
and the majority of the helices are contained within
the boundaries of another helix, forming large
cooperative sets of nested helices. The remaining base
pairs form tertiary interactions that either span two
sets of nested helices, forming a pseudoknot, or are
involved in base triple interactions.
In th*/e
"Matrix of All Possible
Helices" plot
http://www.rna.icmb.utexas.edu/CSI/2STR/, the same RNA
sequence is extended along the X- and Y-axes, with all
potential helices that are comprised of at least four
consecutive Watson-Crick (G:C and A:U) or G:U base
pairs shown below the diagonal line. The helices in the
present comparative structure model are shown above
this line. The number of potential helices is larger
than the actual number present in the
biologically-active structure (see CRW Methods
http://www.rna.icmb.utexas.edu/METHODS/). For example,
the
S. cerevisiae phenylalanine tRNA
sequence, with a length of 76 nucleotides, has 37
possible helices (as defined above); only four of these
are in the crystal structure. The
E. coli 16S rRNA, with 1542
nucleotides (nt), has nearly 15,000 possible helices;
only about 60 of these are in the crystal structure.
For the
E. coli 23S rRNA (2904 nt), there
are more than 50,000 possible helices, with
approximately 100 in the crystal structure. The number
of possible secondary structure models is significantly
larger than the number of possible helices, due to the
exponential increase in the number of different
combinations of these helices. The number of different
tRNA secondary structure models is approximately 2.5 ×
10 19; there are approximately 10 393and 10 740possible
structure models for 16S and 23S rRNA, respectively
(see CRW Methods
http://www.rna.icmb.utexas.edu/METHODS/). Covariation
analysis accurately predicted the structures of the 16S
and 23S rRNAs (see above) from this very large number
of structure models.
1B. Evolution of the 16S and 23S rRNA comparative
structure models
An analysis of the evolution of the
Noller-Woese-Gutell comparative structure models for
the 16S and 23S rRNAs is presented here
http://www.rna.icmb.utexas.edu/CSI/EVOLUTION/(H-1B.1).
Our objective is to categorize the improvements in
these covariation-based comparative structure models by
tabulating the presence or absence of every proposed
base pair in each version of the 16S and 23S rRNA
structure models, starting with our first 16S [ 15 ]
and 23S [ 19 ] rRNA models. Every base pair in each of
the structure models was evaluated against the growing
number and diversity of new rRNA sequences. Proposed
base pairs were taken out of the structure model when
the number of sequences without either a covariation or
a G:C, A:U, or G:U base pair was greater than our
allowed minimum threshold; the nucleotide frequencies
for those base pairs are available from the "Lousy
Base-Pair" tables that are discussed in the next
section. New base pairs were proposed when a (new)
significant covariation was identified with our newer
and more sensitive algorithms that were applied to
larger sequence alignments containing more inherent
variation (see CRW Methods
http://www.rna.icmb.utexas.edu/METHODS/for more
detail).
Although other comparative structure models and base
pairs were predicted by other labs, those interactions
are not included in this analysis of the improvements
in our structure models. The four main structure models
for 16S and 23S rRNA are very similar to one another.
The Brimacombe [ 16 20 ] and Strasburg [ 17 21 ]
structure models were determined independently of ours,
while the De Wachter [ 58 79 ] models were adapted from
our earlier structure models and have incorporated some
of the newer interactions proposed here.
This analysis produced two very large tables with
579 proposed 16S rRNA base pairs evaluated against six
versions of the structure model and 1001 23S rRNA base
pairs evaluated against five versions of the structure
model. Some highlights from these detailed tables are
captured in summary tables (Tables 3aand 3b, and
http://www.rna.icmb.utexas.edu/CSI/EVOLUTION/) that
compare the numbers of sequences and base pairs
predicted correctly and incorrectly for each of the
major versions of the 16S and 23S rRNA structure
models. For this analysis, the current structure model
is considered to be the correct structure; thus, values
for comparisons are referenced to the numbers of
sequences and base pairs in the current structure model
(478 base pairs and approximately 7000 sequences for
16S rRNA, and 870 base pairs and approximately 1050
sequences for 23S rRNA). Three sets of 16S and 23S rRNA
secondary structure diagrams were developed to reveal
the improvements between the current model and earlier
versions: 1) changes since the 1996 published structure
models; 2) changes since 1983 (16S rRNA) or 1984 (23S
rRNA); and 3) all previously proposed base pairs that
are not in the most current structure models
(H-1B.2).
An analysis of these tables reveals several major
conclusions from the evolution of the 16S and 23S rRNA
covariation-based structure models. First,
approximately 60% of the 16S and nearly 80% of the 23S
rRNA base pairs predicted in the initial structure
models appear in the current structure models. The
accuracy of these early models, produced from the
analysis of only two well-chosen sequences, is
remarkable. Second, the accuracy, number of secondary
and tertiary structure interactions, and complexity of
the structure models increase as the number and
diversity of sequences increase and the covariation
algorithms are improved. As well, some pairs predicted
in the earlier structure models were removed from
subsequent models due to the large number of exceptions
to the positional covariation at the two paired
positions. Third, the majority of the tertiary
interactions were proposed in the last few versions of
the structure models.
1C. RNA structure definitions
The RNA structure models presented here are composed
of several different basic building blocks (or motifs)
that are described and illustrated at our RNA Structure
Definitions page
http://www.rna.icmb.utexas.edu/CSI/DEFS/(H-1C.1-2). The
nucleotides in a comparative structure model can be
either base paired or unpaired. Base paired nucleotides
can be part of either a secondary structure helix (two
or more consecutive, antiparallel and nested base
pairs) or a tertiary interaction, which is a more
heterogeneous collection of base pair interactions.
These include any non-canonical base pair (not a G:C,
A:U, or G:U;
e.g., U:U), lone or single base
pairs (when both positions in a base pair are not
flanked by two nucleotides that are base paired to one
another), base pairs in a pseudoknot arrangement, and
base triples (a single nucleotide interacting with a
base pair). Each of these base pair categories has a
unique color code in the illustrations on the "RNA
Structure Definitions" page, which provides multiple
examples of each category from the 16S and 23S rRNA
structure models. In contrast to the nucleotides that
are base paired, nucleotides can also be unpaired in
the comparative structure models. Within this category,
they can be within a hairpin loop (nucleotides capping
the end of a helix), internal loop (nucleotides within
two helices), or in a multi-stem loop (nucleotides
within three or more helices).
2. Nucleotide frequency and conservation
information
2A. Nucleotide frequency tabular display
The nucleotide frequency tables appear in two
general presentation modes. In the traditional table,
the nucleotide types are displayed in the columns,
while their frequencies are shown for each alignment in
the rows. The nucleotide frequencies were determined
for single positions, base pairs, and base triples for
a subset of the RNAs in the CRW Site collection
(detailed in Table 1). Single nucleotide frequencies
are available for all individual positions, based upon
the reference sequence, for every RNA in this
collection. Base pair frequencies are presented for a)
all base pairs in the current covariation-based
structure models, b) tentative base pairs predicted
with covariation analysis, and c) base pairs previously
proposed with comparative analysis that are not
included in our current structure models due to a lack
of comparative support from the analysis with our best
covariation methods on our current alignments (named
"Lousy" base pairs). Base triples are interactions
between a base pair and a third unpaired nucleotide;
base triple frequencies are provided for a) base
triples in the current covariation-based structure
models and b) tentative base triples predicted with
covariation analysis.
For each of these frequency tables, the percentages
of each of the nucleotides are determined for multiple
alignments, where the most similar sequences are
organized into the same alignment. For the three rRNAs,
the alignments are partitioned by their phylogenetic
relationships. There is an alignment for the
nuclear-encoded rRNA for each of the three primary
lines of descent ((1) Archaea, (2) Bacteria, and (3)
Eucarya; [ 80 ] ), each of the two Eucarya organelles
(no alignments yet for the 5S rRNA; (4) Chloroplasts
and (5) Mitochondria), and two larger alignments that
include all of the (6) nuclear-encoded rRNA sequences
for the Archaea, Bacteria, and Eucarya, and (7) these
three phylogenetic groups and the two Eucarya
organelles (Table 2).
For the tRNA and group I and II intron sequences,
the most similar sequences are not necessarily from
similar phylogenetic groups. Instead, the sequences
that are most similar with one another are members of
the same functional and/or structural class. The tRNA
sequences are grouped according to the amino acids that
are bound to the tRNA. Currently, only the type I tRNAs
[ 81 ] are included here; the tRNAs are collected in 19
functional subgroup alignments and one total type I
alignment. The group I and II intron alignments are
based on the structural classifications determined by
Michel (group I [ 32 ] and group II [ 34 ] ) and Suh
(group IE [ 82 ] ). The group I introns are split into
seven alignments: A, B, Cl-2, C3, D, E, and unknown.
The group II introns are divided into the two major
subgroups, IIA and IIB (Table 2).
For the standard nucleotide frequency tables
(Highlight 2A (H-2A)), the left frame in the main frame
window ("List Frame") contains the position numbers for
the three types of tables: single bases, base pairs,
and base triples. Clicking on a position, base pair, or
base triple number will bring the detailed nucleotide
occurrence and frequency information to the main window
("Data Frame;" H-2A.1). The collective scoring data
(H-2A.2) used to predict the base pair is obtained,
where available, by clicking the "Collective Score"
link on the right-hand side of the base pair frequency
table.
As discussed in Section 1A, we have established a
confidence rating for the base pairs predicted with the
covariation analysis; a detailed explanation of the
covariation analysis methods and the confidence rating
system will be available in the Methods section of the
CRW Site http://www.rna.icmb.utexas.edu/METHODS/. The
extent of base pair types and their mutual exchange
pattern (
e.g., A:U <-> G:C) is
indicative of the covariation score. This value
increases to the maximum score as the percentage and
the amount of pure covariations (simultaneous changes
at both positions) increase in parallel with a decrease
in the number of single uncompensated changes, and the
number of times these coordinated variations occur
during the evolution of that RNA (for the rRNAs, the
number of times this covariation occurs in the
phylogenetic tree) increases. These scores are
proportional to our confidence in the accuracy of the
predicted base pair. Red, our highest confidence
rating, denotes base pairs with the highest scores and
with at least a few phylogenetic events (changes at
both paired positions during the evolution of that base
pair). The colors green, black, and grey denote base
pairs with a G:C, A:U, and/or G:U in at least 80% of
the sequences and within a potential helix that
contains at least one red base pair. Base pairs with a
green confidence rating have a good covariation score
although not as high as (or with the confidence of) a
red base pair. Black base pairs have a lower
covariation score, while grey base pairs are invariant,
or nearly so, in 98% of the sequences. Finally, blue
base pairs do not satisfy these constraints;
nevertheless, we are confident of their authenticity
due to a significant number of covariations within the
sequences in a subset of the phylogenetic tree or are
an invariant G:C or A:U pairings in close proximity to
the end of a helix.
The covariation score for each base pair is
determined independently for each alignment (
e.g., Three Domain/Two Organelle,
Three Domain, Archaea,
etc. ). The collective score for
each base pair is equivalent to the highest ranking
score for any one of the alignments. For example, we
have assigned our highest confidence rating to the
927:1390 base pair in 16S rRNA (Figure 2C; H-2A). Note
that the entry for the 927:1390 base pair (H-2A) in the
list of base pairs in the left frame is red in the C
(or confidence) column. For this base pair, only the T
(Three Phylogenetic Domains/Two Organelle) alignment
has a significant covariation score (H-2A); thus, only
the "T" alignment name is red. Of the nearly 6000
sequences in the T alignment, 69% of the sequences have
a G:U base pair, A:U base pair at 16.2%, U:A at 6.9%,
and less than 1% of the sequences have a G:C, C:G, U:U,
or G:G base pair (H-2A.1). The collective scoring data
(H-2A.2) reveals that there are 11 phylogenetic events
(PE) for the T alignment, while the C1+C3 score is
1.00, greater than the minimum value for this RNA and
this alignment (a more complete explanation of the
collective scoring method is available at CRW Methods
http://www.rna.icmb.utexas.edu/METHODS/). Note that the
928:1389 and 929:1388 base pairs are also both red.
Here, six of the seven alignments have significant
extents of covariation for both base pairs and are thus
red. Each of the red alignments have at least two base
pair types (
e.g., G:C and A:U) that occur
frequently, at least three phylogenetic events, and
C1+C3 scores >= 1.5.
2B. Nucleotide frequency mapped onto a
phylogenetic tree
The second presentation mode maps the same
nucleotide frequency data in the previous section onto
the NCBI phylogenetic tree
http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/
[ 60 61 ] (see Materials and Methods for details). This
display allows the user to navigate through the
phylogenetic tree and observe the nucleotide
frequencies for any node and all of the branches off of
that node. The number of nucleotide substitutions on
each branch are displayed, with the number of mutual
changes displayed for the base pairs and base triples.
Currently, only the 16S and 23S rRNA nucleotide
frequencies available in the first tabular presentation
format are mapped onto the phylogenetic tree (see Table
1). As shown in CRW Section 2B (H-2B), the left frame
in the main frame window contains the position numbers
for the three types of data, single bases, base pairs,
and base triples. Clicking on a position, base pair, or
base triple number will initially reveal, in the larger
section of the main frame, the root of the phylogenetic
tree, with the frequencies for the selected single
base, base pair, or base triple. The presentation for
single bases (H-2B.1) reveals the nucleotides and their
frequencies for all sequences at the root level,
followed by the nucleotides and their frequencies for
the Archaea, Bacteria, and Eukaryota (nuclear,
mitochondrial, and chloroplast). Nucleotides that occur
in less than 2%, 1.5%, 1%, 0.5%, 0.2%, and 0.1% of the
sequences can be eliminated from the screen by changing
the green "percentage limit" selection at the top of
the main frame. The number of phylogenetic levels
displayed on the screen can also be modulated with the
yellow phylogenetic level button at the top of the main
frame. Highlight 2B.1 displays only one level of the
phylogenetic tree from the point of origin, which is
the root level for this example. In contrast, Highlight
2B.2 displays four levels from the root. The number of
single nucleotide changes on each branch of the
phylogenetic tree is shown at the end of the row. For
single bases, this number is in black. For base pairs,
there are two numbers. The orange color refers to the
number of changes at one of the two positions, while
the pink color refers to the number of mutual changes
(or covariations) that has occurred on that branch of
the tree (H-2B.2). For example, for the 16S rRNA base
pair 501:544, there are 65 mutual and 74 single changes
in total for the Archaea, Bacteria, Eucarya nuclear,
mitochondrial, and chloroplast. Within the Archaea,
there are six mutual and five single changes. Five of
these mutual changes are within the Euryarchaeota, and
four of these are within the Halobacteriales (H-2B.2).
The base pair types that result from a mutual change
(or strict covariation) are marked with an asterisk
("*").
2C. Secondary structure conservation
diagrams
Conservation secondary structure diagrams summarize
nucleotide frequency data by revealing the nucleotides
present at the most conserved positions and the
positions that are present in nearly all sequences in
the analyzed data set. The conservation information is
overlaid on a secondary structure diagram from a
sequence that is representative of the chosen group (
e.g., E. coli for the gamma
subdivision of the Proteobacteria, or
S. cerevisiae for the Fungi;
H-2C.1). All positions that are present in less than
95% of the sequences studied are considered variable,
hidden from view, and replaced by arcs. These regions
are labeled to show the minimum and maximum numbers of
nucleotides present in that region in the group under
study (
e.g., [0-179] indicates that all
sequences in the group contain a minimum of zero
nucleotides but not more than 179 nucleotides in a
particular variable region). The remaining positions,
which are present in at least 95% of the sequences, are
separated into four groups (H-2C.1): 1) those which are
conserved in 98-100% of the sequences in the group
(shown with red upper-case letters indicating the
conserved nucleotide); 2) those which are conserved in
90-98% of the sequences in the group (shown with red
lower-case letters indicating the conserved
nucleotide); 3) those which are conserved in 80-90% of
the sequences in the group (shown with large closed
circles); and 4) those which are conserved in less than
80% of the sequences in the group (shown with small
open circles).
Insertions relative to the reference sequence are
identified with a blue line to the nucleotides between
which the insertion occurs, and text in small blue font
denoting the maximum number of nucleotides that are
inserted and the percentage of the sequences with any
length insertion at that place in the conservation
secondary structure diagram (H-2C.1). All insertions
greater than five nucleotides are tabulated, in
addition to insertions of one to four nucleotides that
occur in more than 10% of the sequences analyzed for
that conservation diagram. Each diagram contains the
full NCBI phylogenetic classification
http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/for
the group.
Currently, there are conservation diagrams for the
5S, 16S, and 23S rRNA for the broadest phylogenetic
groups: (1) the three major phylogenetic groups and the
two Eucarya organelles, chloroplasts and mitochondria;
(2) the three major phylogenetic groups; (3) the
Archaea; (4) the Bacteria; (5) the Eucarya (nuclear
encoded); (6) the chloroplasts; and (7) the
mitochondria. Longer term, our goal is to generate rRNA
conservation diagrams for all branches of the
phylogenetic tree that contain a significant number of
sequences. Toward this end, we have generated 5S, 16S,
and 23S rRNA conservation diagrams for many of the
major phylogenetic groups within the Bacterial lineage
(
e.g., Firmicutes and
Proteobacteria). We will also be generating
conservation diagrams for the group I and II
introns.
The CRW Site conservation diagram interface (H-2C.2)
provides both the conservation diagrams (in PostScript
and PDF formats) and useful auxiliary information. The
display is sorted phylogenetically, with each row of
the table containing all available conservation
information for the rRNA sequences in that phylogenetic
group. For each of the three rRNA molecules (5S, 16S,
and 23S), three items are available: 1) the reference
structure diagram, upon which the conservation
information is overlaid; 2) the conservation diagram
itself; and 3) the number of sequences summarized in
the conservation diagram, which links to a
web-formatted list of those sequences. The lists, for
each sequence, contain: 1) organism name (NCBI
scientific name); 2) GenBank accession number; 3) cell
location; 4) RNA Type; 5) RNA Class; and 6) NCBI
phylogeny. Users who want more information about a
given sequence should consult the CRW RDBMS (see
below). An equivalent presentation for intron
conservation data is under development.
3. Sequence and structure data
Structure-based alignments and phylogenetic
analysis of RNA structure
Analysis of the patterns of sequence conservation
and variation present in RNA sequence alignments can
reveal phylogenetic relationships and be utilized to
predict RNA structure. The accuracy of the phylogenetic
tree and the predicted RNA structure is directly
dependent on the proper juxtapositioning of the
sequences in the alignment. These alignments are an
attempt to approximate the best juxtapositioning of
sequences that represent similar placement of
nucleotides in their three-dimensional structure. For
sequences that are very similar, the proper
juxtapositioning or alignment of sequences can be
achieved simply by aligning the obviously similar or
identical subsequences with one another. However, when
there is a significant amount of variation between the
sequences, it is not possible to align sequences
accurately or with confidence based on sequence
information alone. For these situations, we can
juxtapose those sequences that form the same secondary
and tertiary structure by aligning the positions that
form the same components of the similar structure
elements (
e.g., align the positions that
form the base of the helix, the hairpin loop,
etc. ). Given the accurate
prediction of the 16S and 23S rRNA secondary structures
from the analysis of the alignments we assembled, we
are now even more confident in the accuracy of the
positioning of the sequence positions in our
alignments, and the process we utilize to build
them.
Aligning new sequences
At this stage in our development of the sequence
alignments, there are well-established and distinct
patterns of sequence conservation and variation. From
the base of the phylogenetic tree, we observe regions
that are conserved in all of the rRNA sequences that
span the three phylogenetic domains and the two
eucaryotic organelles, the chloroplast and
mitochondria. Other regions of the rRNA are conserved
within the three phylogenetic domains although variable
in the mitochondria. As we proceed into the
phylogenetic tree, we observe positions that are
conserved within one phylogenetic group and different
at the same level in the other phylogenetic groups. For
example, Bacterial rRNAs have positions that are
conserved within all members of their group, but
different from the Archaea and the Eucarya
(nuclear-encoded). These types of patterns of
conservation and variation transcend all levels of the
phylogenetic tree and result in features in the rRNA
sequences and structures that are characteristic for
each of the phylogenetic groups at each level of the
phylogenetic tree (
e.g., level one: Bacterial,
Archaea, Eucarya; level two: Crenarchaeota,
Euryarchaeota in the Archaea; level three: gamma,
alpha, beta, and delta/epsilon subdivisions in the
Proteobacteria). Carl Woese likened the different rates
of evolution at the positions in the rRNA to the hands
on a clock [ 4 ] . The highly variable regions are
associated with the second hand; these can change many
times for each single change that occurs in the regions
associated with the minute hand. Accordingly, the
minute hand regions change many times for each single
change in the hour hand regions of the rRNAs. In
addition to the different rates of evolution, many of
the positions in the rRNA are dependent on one another.
The simplest of the dependencies, positional
covariation, is the basis for the prediction of the
same RNA structure from similar RNA sequences (see
Section 1A, Covariation Analysis).
We utilize these underlying dynamics in the
evolution and positional dependency of the RNA to
facilitate the alignment and structural analysis of the
RNA sequences. Our current RNA data sets contain a very
large and diverse set of sequences that represent all
sections of the major phylogenetic branches on the tree
of life. This data collection also contains many
structural variations, in addition to their conserved
sequence and structure core. The majority of the new
RNA sequences are very similar to at least one sequence
that has already been aligned for maximum sequence and
structure similarity; thus, these sequences are
relatively simple to align. However, some of the new
sequences contain subsequences that cannot be aligned
with any of the previously aligned sequences, due to
the excessive variation in these hypervariable regions.
For these sequences, the majority of the sequence can
be readily aligned with the more conserved elements,
followed by a manual, visual analysis of the
hypervariable regions. To align these hypervariable
regions with more confidence, we usually need several
more sequences with significant similarity in these
regions that will allow us to identify positional
covariation and subsequently to predict a new
structural element. Thus, at this stage in the
development of the alignments, the most conserved
regions (
i.e., hour hand regions) and
semi-conserved regions (
i.e., minute hand regions) have
been aligned with high confidence. The second and
sub-second (
i.e., tenth and hundredth of a
second) hand regions have been aligned for many of the
sequences on the branches at the ends on the
phylogenetic tree. However, regions of the sequences
continue to challenge us. For example, the 545 and 1707
regions (
E. coli numbering) contain an
excessive amount of variation in the Eucarya
nuclear-encoded 23S-like rRNAs. These two regions could
not be well aligned and we could not predict a common
structure with comparative analysis with ten Eucaryotic
sequences in 1988 (see Figures 35-43 in [ 48 ] ).
However, once a larger number of related Eucaryotic
23S-like rRNA sequences was determined, we reanalyzed
these two regions and were able to align those regions
to other related organisms (
e.g., S. cerevisiae with
Schizosaccharomyces pombe,
Cryptococcus neoformans, Pneumocystis carinii, Candida
albicans, and
Mucor racemosus ) and predict a
secondary structure that is common for all of these
rRNAs (see Figures 3and 6 in [ 52 ] ). While the
secondary structures for the fungal 23S-like rRNAs are
determined in these regions, the animal rRNAs were only
partially solved. We still need to determine a common
secondary structure for the large variable-sized
insertions in the animal rRNAs, and this will require
even more animal 23S-like rRNA sequences from organisms
that are very closely related to the organisms for
which we currently have sequences.
A large sampling of secondary structure
diagrams
We have generated secondary structure diagrams for
sequences that represent the major phylogenetic groups,
and for those sequences that reveal the major forms of
sequence and structure conservation and variation. New
secondary structure diagrams are templated from an
existing secondary structure diagram and the alignment
of these two sequences, the sequence for the new
structure diagram and the sequence for the structure
that has been templated. The nucleotides in the new
sequence replace the templated sequence when they are
in the same position in the alignment, while positions
in the new sequence that are not juxtaposed with a
nucleotide in the templated sequence are initially left
unstructured. These nucleotides are then placed
interactively into their correct location in the
structure diagram with the program XRNA (Weiser &
Noller, University of California, Santa Cruz) and
base-paired when there is comparative support for that
pairing in the alignment; otherwise, they are left
unpaired.
The process of generating these secondary structure
diagrams occurs in parallel with the development of the
sequence alignments. In some cases, the generation of a
structure diagram helps us identify problems with the
sequence or its alignment. For example, anomalies in
structural elements (in the new structure diagram) that
had strong comparative support in the other sequences
could be the result of a bad sequence or due to the
misalignment of sequences in the helix region. In other
cases, the new structure diagram reveals a possible
helix in a variable region that was weakly predicted
with comparative analysis. However, a re-inspection of
a few related structure diagrams revealed another
potential helix in this region that was then
substantiated from an analysis of the corresponding
region of the alignment. Thus, the process of
generating additional secondary structure diagrams
improves the sequence alignments and the predicted
structures, in addition to the original purpose for
these diagrams, to reveal the breadth of sequence
conservation and variation for any one RNA type.
Our goals for the "Sequence and Structure Data"
section of the CRW Site are to:
A) Align all rRNA, group I and II intron sequences
that are greater than 90% complete and are available at
GenBank;
B) Generate rRNA and group I/II intron secondary
structure diagrams for organisms that are
representative of a phylogenetic group or
representative of a type of RNA structural element. The
generation of 5S, 16S, and 23S rRNAs secondary
structures from genomic sequences generally has higher
priority over other rRNA sequences.
C) Enter pertinent information for each sequence and
structure into our relational database management
system. This computer system organizes all of our RNA
sequence and structure entries, associates them with
the organisms' complete NCBI phylogeny
http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/,
and allows for the efficient retrieval of this data
(see Section 4: Data Access Systems for more
details).
Due in part to the technological improvements in the
determination of nucleic acid sequence information, the
number of ribosomal RNA and group I and II intron
sequences has increased significantly within the past
10 years. As of December 2001, the approximate numbers
of complete or nearly complete sequences and secondary
structure diagrams for each of these RNAs for the major
phylogenetic groups and structural categories are shown
in Highlight 3A.1. At this time, the actual number of
sequences that are both greater than 90% complete and
available at GenBank is greater than the number in our
CRW RDBMS.
The sequences, alignments, and secondary structure
diagrams are available from several different web
pages, which are described below in Sections 3A-3D and
4A-4B.
3A. Index of available sequences and
structures
The top section of the "Index of Available Sequences
and Structures" page (H-3A.1) reveals the numbers of
available sequences for the Archaea, Bacteria, and
Eucarya nuclear, mitochondrial, and chloroplast groups
that are at least 90% complete and structure diagrams
for the 5S, 16S, and 23S rRNAs and group I and group II
introns. The remainder of the index page contains the
numbers of sequences and structures for more expanded
lists for each of those five phylogenetic/cell location
groups. For example, the Archaea are expanded to the
Crenarchaeota, Euryarchaeota, Korarchaeota, and
unclassified Archaea. These counts are updated
dynamically when the information in our relational
database management system is revised. The numbers of
sequences and structures are links that open the RDBMS
"standard" output view (see below for details) for the
selected target set. Secondary structure diagrams are
available in PostScript, PDF, and BPSEQ (see above)
formats from the structure links. The organism names in
the output from these links are sorted alphabetically.
The number of entries per output page is selectable
(20, 50, 100, 200, or 400), with 20 set as a default.
Entries not shown on the first page can be viewed by
clicking on the "Next" button at the bottom left of the
output page.
As of December 2001, our data collection contains
11,464 rRNA (5S, 16S, and 23S) and intron (group I, II,
and other) sequences. The ribosomal RNAs comprise 80%
of this total, and 16S rRNA represents 82% of the rRNA
total; the remainder is split between the 23S and 5S
rRNAs. Intron sequences comprise 20% of our total
collection, with approximately twice as many group I
introns than group II introns. Of the 406 secondary
structure diagrams, the majority are for the 16S (71%)
and 23S (20%) rRNAs. At this time, tRNA records are not
maintained in our database system.
3B. New secondary structure diagrams
Secondary structure diagrams that have been created
or modified recently are listed and available from
their own page (H-3B.1). These diagrams are sorted into
one of three categories: new or modified 1) in the past
seven days (highlighted with red text); 2) in the past
month (blue text); and 3) in the past three months
(black text). Diagrams are listed alphabetically by
organism name within each of the three time categories.
The display also indicates the cell location and RNA
Class (see below) for each diagram. The PostScript,
PDF, and BPSEQ files can be viewed by clicking the
appropriate radio button at the top of this page and
then the links in the structure field.
3C. Secondary structure diagram retrieval
Multiple secondary structure diagrams can be
downloaded from the Secondary Structure Retrieval Page
(Highlight 3C.1). This system allows the user to select
from organism names, phylogeny (general: Archaea,
Bacteria, Eukaryota, and Virus), RNA Class (see Table
4), and cell location, as well as selecting for
PostScript, PDF, or BPSEQ display formats. Once these
selections are made, a list of the structure diagrams
that fit those criteria appears. The user may select
any or all of the diagrams to be downloaded. The 23S
rRNA diagrams (which appear in two halves) are
presented on one line as a single unit to ensure that
both halves are downloaded. The system packages the
secondary structure diagrams files into a compressed
tar file, which can be uncompressed with appropriate
software on Macintosh, Windows, and Unix computer
platforms. (Note: due to a limitation in the web server
software, it is currently not possible to reliably
download more than 300 structures at one time. This
limitation can be avoided by subdividing large
queries.)
3D. Sequence alignment retrieval
The Sequence Alignment Retrieval page (Highlight
3D.1) provides access to the sequence alignments used
in the analyses presented at the CRW Site. Sequence
alignments are available in GenBank and AE2 (Macke)
formats (Table 2). These alignments will be updated
periodically when the number of new sequences is
significant. Newer alignments might also contain
refinements in the alignments of the sequences. For
each alignment, there is a corresponding list of
sequences, their phylogenetic placement, and other
information about the sequences (see conservation list
of sequences for conservation diagrams). At present,
only the rRNA alignments are available; the group I and
group II intron alignments will be made available in
June 2002.
3E. rRNA Introns
The introns that occur in 16S and 23S rRNAs are
organized into four preconfigured online tables. These
tables disseminate the intron information and emphasize
the major dimensions inherent in this data: 1) intron
position in the rRNA, 2) intron type, 3) phylogenetic
distribution, and 4) number of introns per exon
gene.
3E. rRNA Introns Table 1: Intron Position
The introns in rRNA Introns Table 1 are organized by
their position numbers in the 16S and 23S rRNAs. The
16S and 23S rRNA position numbers are based on the
E. coli rRNA reference sequence
(J01695) (see Table 1). The intron occurs between the
position number listed and the following position (
e.g., the introns between
position 516 and 517 are listed as 516). rRNA Introns
Table 1 has four components.
The total number of introns and the number of
positions with at least one intron in 16S and 23S rRNA
are shown in rRNA Introns Table 1A (see highlights
below and H-3E.1). The list of all publicly available
rRNA introns, sorted by the numeric order of the intron
positions, is contained in rRNA Introns Table 1B. This
table has nine fields: 1) rRNA type (16S or 23S); 2)
the intron position; 3) the number of documented
introns occurring at that position; 4) the intron types
(RNA classes) for each rRNA intron position; 5) the
number of introns for each intron type for each rRNA
position; 6) the length variation (minimum # - maximum
#) for introns in each intron type; 7) the cell
location for each intron type; 8) the number of
phylogenetic groups for each intron type, (here,
defined using the third column from rRNA Introns Table
3: Phylogenetic Distribution); and 9) the organism name
and accession number.
These fields in rRNA Introns Table 1B (H-3E.1) allow
for a natural dissemination of the introns that occur
at each rRNA site. For example, of the 116 introns (as
of December 2001) at position 516 in 16S rRNA, 55 of
them are in the IC1 subgroup (H-3E.2); these introns
range from 334-1789 nucleotides in length, all occur in
the nucleus, and are distributed into four distinct
phylogenetic groups. 54 of the introns at position 516
are in the IE subgroup, range from 190-622 nucleotides
in length, all occur in the nucleus, and are also
distributed into four distinct phylogenetic groups,
etc.
Additional information is available in a new window
for each of the values in rRNA Introns Table 1B
(H-3E.3). This information is retrieved from the
relational database management system (see section 4).
The information for each intron entry in the new window
are: 1) exon (16S or 23S rRNA); 2) intron position in
the rRNA; 3) intron type (RNA class); 4) length of
intron (in nucleotides); 5) cell location; 6) NCBI
phylogeny; 7) organism name; 8) accession number; 9)
link to structure diagram (if it is available); and 10)
comment.
The number of intron types per intron position are
tabulated in rRNA Introns Table 1C (H-3E.4), while the
number of introns at each rRNA position are ranked in
rRNA Introns Table 1D (H-3E.5). This latter table
contains six fields of information for each rRNA: 1)
number of introns per rRNA position; 2) number of
positions with that number of introns; 3) the rRNA
position numbers; 4) total number of introns (field #1
× field #2); 5) the Poisson probability (see rRNA
Introns Table 1D for details); and 6) the expected
number of introns for each of the observed number of
introns per rRNA site.
The highlights from rRNA Introns Table 1 are: 1) As
of December 2001, there are 1184 publicly available
introns that occur in the rRNAs, with 900 in the 16S
rRNA, and 284 in 23S rRNA. These introns are
distributed over 152 different positions, 84 in the 16S
rRNA and 68 in 23S rRNA. 2) Although 16S rRNA is
approximately half the length of 23S rRNA, there are
more than three times as many introns in 16S rRNA.
However, this bias is due, at least in part, to the
more prevalent sampling of 16S and 16S-like rRNAs for
introns. 3) The sampling of introns at the intron
positions is not evenly distributed (1184/152 = 7.79
introns per position for a random sampling). Instead,
nearly 50% (71/152) of the intron positions contain a
single intron and 89% (135/152) of the intron positions
contain ten or less introns. In contrast, 59%
(681/1163) of the introns are located at 9% of the
intron positions and the three intron positions with
the most introns (943, 516, and 1516 in 16S rRNA)
contain 361, or 31% (361/1163), of the rRNA introns. 4)
rRNA Introns Table 1D compares the observed
distribution of rRNA introns with the Poisson
distribution for the observed number of introns. The
Poisson distribution,
P(x) =
e -μμ
x
x! -1, where μ is the mean
frequency of introns for positions in a particular exon
and
x is the target number of introns
present at a particular position, allows the
calculation of expected numbers of positions containing
a particular number of introns. Based upon the observed
raw numbers of introns in the 16S and 23S rRNAs, we
expect to see no positions in 16S rRNA containing more
than five introns and no positions in 23S rRNA
containing more than three introns. However,
thirty-five rRNA positions fall into one of those two
categories. We also see both more positions without
introns and fewer positions containing only one or two
introns than expected. This observed distribution of
rRNA introns among the available insertion positions is
extremely unlikely to occur by chance. 5) While a
single intron type occurs at the majority of the intron
positions, several positions have more than one intron
type. A few of the positions that deserve special
attention have IC1 and IE introns at the same position
(16S rRNA positions 516 and 1199, and 23S rRNA position
2563). The 16S rRNA position 788 has several examples
each of IC1, IIB, and I introns.
3E. rRNA Introns Table 2: Intron Type
The introns are organized by intron type, as defined
above, in rRNA Introns Table 2 (H-3E.6). The frequency
of 16S and 23S rRNA exons, non-rRNA exons, number of
intron positions in the 16S and 23S rRNA, cell
locations, and number of phylogenetic groups for each
intron type are tabulated. The highlights of this table
are: 1) Of the 1184 known rRNA introns, 980 (83%) are
group I, 21 (2%) are group II introns, and the
remaining 183 (15%) are unclassified (see below). While
only 2% of the rRNA introns are group II, 62%
(728/1180) of the non-rRNA introns are group II. In
addition to the group II introns, nearly all of the IC3
introns do not occur in rRNAs. 2) The majority of the
rRNA group I introns (851/980 = 87%) fall into one of
three subgroups: I (276 introns), IC1 (415 introns),
and IE (160 introns). 3) As noted earlier, there are
three times as many 16S rRNA group I introns than 23S
rRNA group I introns (753 vs. 227). 4) Among the three
cellular organelles in eucaryotes, 1010 introns (85%)
occur in the nucleus, 133 (11%) in the mitochondria,
and 41 (4%) in the chloroplasts. 5) The subgroups IC1,
IC3 and IE are only present in the nucleus, while the
IA, IB, IC2, ID, and II subgroups occur almost
exclusively in chloroplasts and/or mitochondria.
The 183 introns described in rRNA Introns Table 2 as
"Unclassified" merit special attention. All of these
introns do not fall into either the group I and group
II categories; however, two notable groups of introns
are included within the "Unclassified" category. The
first is a series of 43 introns occurring in Archaeal
rRNAs (the Archaeal introns). Thirty-one of the known
Archaeal introns are found in 16S rRNA and the
remaining twelve are from 23S rRNA exons. The Archaeal
introns range in length from 24 to 764 nucleotides,
with an average length of 327 nucleotides. The second
group contains 121 spliceosomal introns found in fungal
rRNAs. 92 spliceosomal introns are from 16S rRNA and 29
are from 23S rRNA; the lengths of these introns range
from 49 to 292 nucleotides. A future version of this
database will include both of these groups as separate,
distinct entries. Both the Archaeal and splicesomal
introns occur only in nuclear rRNA genes and tend to
occur at unique sites; the lone exception is the
spliceosomal intron from
Dibaeis baeomyces nuclear 23S
rRNA position 787, a position where a group IIB intron
occurs in mitochondrial
Marchantia polymorpha rRNA. The
Unclassified group contains 21 introns that do not fall
into any of the four previously discussed categories
(group I, group II, Archaeal, or spliceosomal),
including all four mitochondrial introns in this
group.
rRNA Introns Table 2 expands the presentation by
providing links to twenty additional tables (H-3E.7),
each of which provides expanded information about a
specific intron type. The organism name, exon, intron
position, cell location, and complete phylogeny are
accessible for each intron from these tables. These
online tables are dynamically updated daily as
information about new introns is made available.
3E. rRNA Introns Table 3: Phylogenetic
Distribution
The distribution of introns on the phylogenetic tree
is tabulated in rRNA Introns Table 3A (H-3E.8) and 3B
(H-3E.9). rRNA Introns Table 3A reveals the ratio of
the number of rRNA introns per rRNA gene for the
nuclear, chloroplast, and mitochondrial encoded RNAs
for the major phylogenetic groups. The most noteworthy
distributions are: 1) The majority (96%) of the rRNA
introns occur in Eucarya, followed by the Archaea, and
the Bacteria. 2) Only one rRNA intron has been
documented in the Bacteria; due to the large number of
rRNA gene sequences that have been determined, the
ratio of rRNA introns per rRNA gene is essentially zero
for the bacteria. 3) The frequency of introns in
Archaea rRNAs is higher, with 43 examples documented as
of December 2001. Within the Archaea, there is a higher
ratio of rRNA introns in the Desulfurococcales and
Thermoproteales subbranches in the Crenarchaeota
branch. 4) For the three primary phylogenetic groups,
the highest ratio of rRNA introns per rRNA gene is for
the Eucarya, and for the phylogenetic groups within the
Eucarya that have significant numbers of rRNA
sequences, the ratio is highest in the fungi. Here, the
ratios of rRNA introns per rRNA gene are similar
between the nucleus and mitochondria (1.34 for the
nucleus, 1.20 for the mitochondria). A significant
number of rRNA introns occurs in the plants, with
similar ratios of rRNA intron/rRNA gene for the
nucleus, chloroplast, and mitochondria (0.36 for the
nucleus, 0.38 for the chloroplast, and 0.34 for the
mitochondria). In sharp contrast with the fungi and
plants, only one intron has been documented in an
animal rRNA, occurring within the
Calliphora vicina nuclear-encoded
23S-like rRNA (GenBank accession number K02309).
Each of the two special "Unclassified" rRNA intron
groups has a specific phylogenetic bias. Archaeal rRNA
introns, which have unique sequence and structural
characteristics [ 83 ] , have not yet been observed
within the Euryarchaeota or Korarchaeota; in fact, no
non-Archaeal introns have been found in Archaea rRNAs
to date. Splicesomal rRNA introns have only been
reported in 31 different genera in the Ascomycota [ 84
] . rRNA Introns Table 3A also presents the numbers of
(complete or nearly so) rRNA sequences in the same
phylogenetic groups in order to address the question of
sampling bias. Two important caveats to this data must
be considered. First, the numbers of rRNA sequences are
an underestimate, since many rRNA introns are published
with only short flanking exon sequences and do not meet
the 90% completeness criterion for inclusion in this
rRNA sequence count. The second caveat is that many
rRNA sequences contain multiple introns (see rRNA
Introns Table 4 and related discussion, below, for more
information). Of the 51 phylogenetic group/cell
location combinations shown in rRNA Introns Table 3
that may contain rRNA introns, 15 (29%) have a
intron:rRNA sequence ratio greater than 1.0, indicating
a bias toward introns within those groups. Introns are
comparatively rare within the 26 (51%) groups that have
a ratio below 0.3; ten of these 26 groups contain no
known rRNA introns. Ten (20%) of the groups have
intermediate ratios (between 0.3 and 1.0).
A more detailed phylogenetic distribution is
available in rRNA Introns Table 3B (H-3E.10). The first
three fields contain levels 2, 3, and 4 of the NCBI
phylogeny, followed by fields for the genus of the
organism, cell location, exon (16S or 23S rRNA), and
intron type. Each of these classifications include a
link to the complete details (organism name, phylogeny,
cell location, exon, intron position, intron number,
accession number, and structure diagram (when
available)) for the intron sequences in that group.
3E. rRNA Introns Table 4: Number of Introns per
Exon
rRNA Introns Table 4 presents the number of introns
per rRNA gene (H-3E.11). While more than 80% of the
documented rRNA genes do not have an intron, 646 16S
and 182 23S rRNAs have at least one intron.
Approximately 75% (623) of these genes have a single
intron, 15% (127) have two introns, 0.5% (40) have
three, 0.25% (20) have four, 0.1% (11) have five, two
rRNA genes have 6, 7 or 8 introns, and one rRNA gene
has 9 introns.
To determine the amount of bias in the distribution
of introns among their exon sequences, the Poisson
distribution (here, μ is the mean frequency of introns
for a particular exon and
x is the target number of introns
per rRNA gene) has been used to calculate the number of
rRNA sequences expected to contain a given number of
introns (rRNA Introns Table 4). Based upon this data,
no rRNA sequences are expected to contain four or more
introns; in fact, we see 38 sequences that contain
these large numbers of introns. The observed numbers of
sequences exceed the expected values for all but one
category: fewer rRNAs contain only one intron than
expected.
The two molecules (16S and 23S rRNA) show a
differing trend with respect to cell location for those
sequences containing large numbers of introns. In 16S
rRNA, only nuclear genes (ten) have been observed to
contain five or more introns; indeed, of the 57 genes
containing three or more introns, only two are not
nuclear (both of these are mitochondrial). In 23S rRNA,
the trend is both opposite and weaker; of the thirteen
rRNA sequences containing four or more introns, five
are nuclear (containing five introns), with four
chloroplast and four mitochondrial genes comprising the
remaining eight sequences.
rRNA Introns Table 4 provides access to seventeen
additional tables (H-3E.12), which each present the
complete information for every intron within a
particular class (
e.g., 16S rRNA genes containing
two introns), grouped by their exons. As with the other
online tables, this information will be updated daily
to reflect new intron sequences that are added to this
database.
The final components of the "rRNA Introns" page are
16S and 23S rRNA secondary structure diagrams that show
the locations for all of the known rRNA introns
(H-3E.13). The information collected here on the "rRNA
Introns" page is the basis for two detailed analyses
that will be published elsewhere: 1) the spatial
distribution of introns on the three dimensional
structure of the 16S and 23S rRNA (Jackson
et al., manuscript in
preparation); and 2) the statistical analysis of the
distribution of introns on the rRNA (Bhattacharya
et al., manuscript in
preparation).
3F. Group 1/11 Intron distributions
For the CRW Site project, we collect group I and II
introns and all other introns that occur in the
ribosomal RNA. The "Intron Distribution Data" page
contains three tables that compare intron types,
phylogeny, exon, and cell location.
Intron Distribution Table 1 maps "Intron Type" vs.
"Phylogeny" (and "Cell Location;" H-3F.1). Group I and
II intron data are highlighted with yellow and blue
backgrounds, respectively. The phylogenetic divisions
are also split into the three possible cellular
locations (nuclear, chloroplast, and mitochondria). A
few of the highlights are:
1) the Eukaryota contain the majority (2218 / 2349 =
94%) of the introns in the CRW RDBMS. 2) The Archaea
have 42 introns that have unique characteristics and
are called "Archaeal introns." 3) Group I introns are
present in eukaryotes (nuclear-, chloroplast-, and
mitochondrial-encoded genes) and in Bacteria. Group II
introns have only been observed in Bacteria and in
Eukaryotic chloroplast and mitochondrial genes.
Intron Distribution Table 2 shows "Intron Type" vs.
"Exon" (and "Cell Location;" H-3F.2). Again, group I
and II intron data are highlighted with yellow and blue
backgrounds, respectively. In this table, the exon
types are split into the three possible cellular
locations (nuclear, chloroplast, and mitochondria). As
of December 2001, the most obvious trend is that the
exons with the most Group I introns are 16S rRNA (900),
leucine tRNA (337), 23S rRNA (284), ribosomal protein
S16 (214), and ribosomal protein L16 (152).
Intron Distribution Table 3 compartmentalizes the
intron data by "Phylogeny" and "Exon" (and "Cell
Location;" H-3F.3). In this table, color is used to
highlight the three phylogenetic domains (Archaea in
yellow, Bacteria in blue, and Eukaryota in green). As
in Intron Distribution Table 2, the exon types are
split into the three possible cellular locations
(nuclear, chloroplast, and mitochondria).
Each of these three tables is dynamically created
from a specific series of RDBMS queries on a daily
basis. As of December 2001, links connecting to the
specific RDBMS results are not available.
4. Data access systems
4A. RDBMS (Standard)
The "Standard" interface is the most fundamental of
our interfaces to the CRW RDBMS information. While the
restricted, specialized interface to the RDBMS
information in Section 3A requires minimal instruction
to use, the standard interface, with its ability to
cull out all arrangements of information from the
different fields with sophisticated search queries and
output field sortings, requires a quick lesson for its
operation. The selection process has three stages: 1)
selection of attribute fields to display; 2)
determination of values for the search; and 3)
adjustment of the output field sort order.
A detailed explanation for each of the attributes is
available from the links to the attribute names. This
information is shown in the right frame. Additional
examples of this system are available online.
Step 1. At the onset, the user selects the fields to
be displayed on the screen and then clicks the "Go"
button. While the user can select the individual fields
(
e.g., "Organism" or "Phylogeny"),
for most applications the "QrRNA" (query rRNA),
"Qintron" (query intron), or "All" options will
automatically click the appropriate fields that are
most important for searching for ribosomal RNA or group
I and II intron entries.
Step 2. Select values for the fields or attributes.
The acceptable values for the attributes in our RDBMS
system are shown on the main frame of the query page
(for list- and button-driven fields) or, for text input
fields, can be determined with the "V" (values) button
on the right side of the main frame; the results are
displayed in the right frame (see Figure 3and
H-4A.1).
• The values for cellular location are Chl
(chloroplast), Cya (cyanelle), Mit (mitochondria), Nuc
(nuclear), and Vir (viral); each can be selected by
simply checking the box to the left of its name.
• The values for the attributes RNA Type, ORF (open
reading frame), Secondary Structures (entries
with/without secondary structure diagrams),
Results/Page, and Color Display are also displayed on
the main frame, and can be selected by clicking the
appropriate box or button.
The values for other attributes such as RNA Class,
Sequence Length, and Exon can be determined by
selecting one or more of the values in the scroll box.
The values for these attributes can also be found by
clicking the "V" button associated with each attribute.
For example, clicking on the "Exon" "V" button will
reveal, in the right frame, all of the exons that are
contained in our database. The same exons are present
in the scroll box.
• The values displayed for any one attribute are
dependent on the settings of the other attributes. For
example, when only rRNA is selected for the "RNA Type,"
then there are no values for "Exon." All of the
possible exon values are displayed when "Intron" is the
selected "RNA Type," while only a subset of the
possible exon values are shown when Mit (mitochondria)
is the selected "Cell Location." Note: no selection for
an attribute signifies to this system that all of the
values are possible.
The values present in our database for the
attributes "Organism," "Phylogeny" (except for the
first level - Archaea, Bacteria, and Eukaryota - that
can be selected from the main frame), "Common Name"
(except for the first level: "Animals,"
"Fungi&Plants," "Protists"), "Accession Number,"
"Intron Position," and "Comment" can only be observed
in the right frame after clicking the "V" button.
• The values selected with the mouse in the right
frame will appear in the appropriate attribute
field.
• The values for each attribute are dependent on the
settings for the other attributes. For example, if
there are many values for the "Organism" field,
selecting Archaea in the "Phylogeny" field will reduce
the number of names in the "Organism" field to just
those that are in this phylogenetic group.
• The number of possible values for an attribute can
also be constrained by entering only part of a value in
the field. For example, typing 'Esch' in the "Organism"
field will output several organism names that contain
'Escherichia' when the "V" button is clicked. Typing
"coli" in this field will list all organism names that
contain "coli," as either part of a name or a complete
word.
• Note that the system is case sensitive for all
fields except "Common Name." The text 'esch' in the
same "Organism" field will not output 'Escherichia' in
the right frame.
The "Phylogeny" field with the values frame on the
right was developed to allow the user to navigate
through the phylogenetic tree. The information for the
"Phylogeny" and "Common Name" fields is downloaded from
the NCBI (see Materials and Methods; this information
is downloaded daily to assure that we have the most
current version of this data). There are two general
modes of operation.
For mode one, you can systematically navigate
through the phylogenetic tree to the selected goal
point. For example, to get to the last phylogenetic
group that contains
Homo sapiens and gorillas, the
user would click on the "Eukaryota" phylogeny button,
then click on the "Fungi/Metazoa group" link in the
right frame, followed by the "Metazoa," "Eumetazoa,"
"Bilateria," "Coelomata," "Deuterostomia," "Chordata,"
"Craniata," "Vertebrata," "Gnathostomata,"
"Teleostomi," "Euteleostomi," "Sarcopterygii,"
"Tetrapoda," "Amniota," "Mammalia," "Theria,"
"Eutheria," "Primates," "Catarrhini," and "Hominidae"
links. The phylogenetic group Hominidae contains the
genera Gorilla, Pan (chimpanzees), Pongo, and Homo (see
Figure 3, H-4A.1, and H-4A.2). This type of navigation
is useful when you know the links that will get you to
the desired goal point; otherwise, mode two can help
you jump to the appropriate node in the phylogenetic
tree.
For the second mode, you type all or part of the
name of an organism or phylogenetic group that is close
to the phylogenetic node you want. For example, type
"Homo sapiens" in the "Phylogeny" field and press the
"V" button in the "Phylogeny" field. The right frame
will display a few names; from these, select "Homo
sapiens." The right frame now contains the entire
phylogenetic path from the base of the tree to Humans
(Figure 3and H-4A.1).
The "Common Name" attribute can also help identify
organism names in the CRW RDBMS. As with the phylogeny
operation, two general modes for determining the values
are available. For the first, the user would type the
presumed common name in the "Common Name" field, and
click the "V" button. A few general examples are: worm,
fish, cat, dog, and human. More specific examples are:
common earthworm (
Lumbricus terrestris ), European
polecat (
Mustela putorius ), and duckbill
platypus (
Ornithorhynchus anatinus ). These
names must be in the "Common Name" database for the
sequence entry to be identified with this method. In
contrast, the second mode is intended to identify
larger groups of organisms. The three buttons in the
"Common Name" field ("Animals," "Fungi&Plants,"
"Protists;" H-4A.3) each reveal various low-level
common names in the right frame that are arranged in a
pseudo-phylogenetic structure. For example, a few of
the lower animals (sponges, flatworms,
etc. ) are listed when the
"Animals" button is pressed, in addition to the
Protostomia, Deuterostomia, and organisms nested within
these groups (Arthropoda, chordates, vertebrates,
Mammals,
etc.; H-4A.3). Accordingly, the
"Fungi&Plants" and "Protists" buttons reveal the
major groups of organisms within their respective
groups. For the latter mode of operation, the user
selects one of these common names, such as "Mammals."
The phylogeny for this group then appears in the same
right frame (cellular organisms, Eukaryota,
Fungi/Metazoa group, Metazoa, Eumetazoa, Bilateria,
Coelomata, Deuterostomia, Chordata, Craniata,
Vertebrata, Gnathostomata, Teleostomi, Euteleostomi,
Sarcopterygii, Tetrapoda, Amniota, Mammalia), along
with the two phylogenetic groups within the Mammals
(Mammalia), Prototheria and Theria. Another example is
the common name "Mosses" in the Fungi&Plants.
Selecting "Mosses" brings up the phylogeny for the
Bryophyta. Note that these common names (
i.e., "mammals" or "mosses") do
not appear in the common name field in the output for
the sequence entries that are within the Mammalian or
Bryophyta phylogenetic groups. Thus, the common name
field could be very useful to identify organisms and
phylogenetically related organisms when you don't know
their genus/species organism name or the phylogeny for
that group of organisms.
Step 3. The last, critical step before submitting a
query is to select the sort order for the attributes in
the output. While a query will yield the same number of
results with any sort order, the choice of sort order
can make answering questions easier. Take, for example,
a search for all Eucarya rRNA entries. By default, the
entries are sorted alphabetically first by their
phylogenetic classification, followed by organism name,
cell location, and last by their RNA class. In
contrast, the sort orders <phylogeny, organism name,
cell location, and RNA class> and <organism name,
RNA class, phylogeny, and cell location> produce
significantly different orders and overall arrangements
for the same set of entries (see online examples); the
second sorting is more useful when searching for a
particular organism, since its exact location on the
phylogenetic tree may not be known to the user. The
output page (H-4A.2) reveals the search strategy and
attribute sort order at the bottom of the page. The
default sort order for the attributes is shown on the
"S" (or sort) buttons on the right side of the main
frame (Figure 3and H-4A.1). The sort order is changed
by simply clicking the "S" buttons in the order the
attributes are to be sorted. The resulting sort order
for the attributes are shown in the small text box to
the left of each attribute's S button; alternatively,
you can type numbers into these boxes to set the sort
order. The alphabetical/numerical order for any
attribute can be reversed (z -> a, high number ->
low number) by checking the box in the "R" (or reverse)
column to the right of the Sort buttons. Finally, the
sortings can be reset to the default values by clicking
the "Sort Reset" button at the top of the query
page.
Before submitting the query, a few attributes
deserve more attention.
• Secondary Structures: a comparative secondary
structure model has been developed for more than 400 of
the sequence entries (see Section 3). The 'secondary
structure' attribute near the bottom of the query page
is an option to output
all sequence and structure
entries, only those entries
with a secondary structure, or
entries
without a secondary structure
diagram.
• Results/Page: the number of entries per output
page can be modulated. While the system defaults to 50
entries per page, the maximum number of entries per
output page can be set to 20, 100, 200, and 400. The
user can scroll to those entries that do not appear on
the first page by selecting the "Next" button on the
left bottom frame in the output window and use the
"Previous" button in the same frame to move toward the
first page, as necessary.
• Color Display: to help distinguish the organism
names on the output pages, the entries have the same
color when the organism names are the same. The colors
(pink and white) alternate for changes in the organism
names in the output entries.
• Group ID and Group Class: these two attributes are
currently not fully functional; thus, we do not
encourage their use at this time.
• RNA Type/Class: currently, we do not have data
entries for the following RNA Types and Classes: mRNA,
tRNA, SnRNA, and Other.
After clicking the submit button at the top or
bottom of the query page, a new window will open. This
window distributes the results into three frames
(H-4A.2). The main frame contains the sequence and
structure entries that satisfy the search query. The
frame in the lower left indicates the number of entries
shown in the window and the entry numbers currently
shown, and, if necessary, contains buttons to scroll to
the next or previous set of entries. The third frame at
the bottom middle-right displays the total number of
entries that satisfy the query, the search strategy and
the sort order for this query.
The three formats for the secondary structure
diagrams, PostScript, PDF, and BPSEQ (see Section 1A
and the online help from the "Secondary Structure" and
"StrDiags" links on the RDBMS query and results pages)
can be retrieved from the results window. The system
defaults to PostScript when the secondary structure
link is clicked; PDF or BPSEQ files can be obtained
instead from the structure link by selecting the
corresponding radio button at the top left section of
the main frame. An explanation of the structure link
names (d.5, d.l6, d.235, d.233, b.Il, and a.I2) and the
longer names that are associated with the downloaded
structure files is also available online.
The GenBank accession number for each entry is a
link to a new window that retrieves the specified entry
from NCBI. Sequence entries with more than one GenBank
number contain a "m" to the right of the accession
number. Clicking the "m" link opens a new window with
all of the GenBank numbers associated with this
sequence.
Each entry is associated with a NCBI phylogeny
listing that can be retrieved in a new window by
clicking the "m" button in the Phylogeny column. This
listing also contains the known common names associated
with each level of the phylogenetic tree (H-4A.4). The
phylogeny for all of the entries in the results window
is available in a new window when the "M" button in the
header line of the phylogeny field is clicked.
4B. RDBMS (PhyloBrowser)
The PhyloBrowser interface to the CRW RDBMS was
developed to facilitate the identification and
retrieval of sequence and structure entries that are
associated with specific phylogenetic groups. While the
Standard interface will reveal all sequence entries for
any one phylogenetic group, it does not show the
phylogenetic groups that do not have the requested
sequences; the PhyloBrowser interface displays the
entire phylogenetic tree, including those branches that
do not have corresponding entries. This interface is
based on the Taxonomy Browser developed by NCBI
http://www.ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html/and
uses the NCBI taxonomy database [ 60 61 ] . Here, we
describe the PhyloBrowser interface, ways to navigate
through the phylogenetic data, and how to retrieve RNA
information using this system.
The PhyloBrowser uses three frames (Figure 4and
H-4B.1). At the bottom of the page is the Results Frame
(white background), which displays the selected portion
of the phylogenetic tree and any RNA information. In
the upper left is the Selection Frame (pink
background), where the user can select the phylogenetic
and RNA information shown in the Results Frame. Help is
provided in the Help Frame, at the upper right (blue
background).
Starting at the root, the entire phylogenetic tree
can be navigated with this system. The base
phylogenetic level name is shown in green. The number
of phylogenetic levels displayed (below the base level)
can be modulated from one (the default) to five levels
using the "Display Phylogenetic Levels" control in the
Selection Frame. The phylogenetic level number for each
group is shown in red preceding the phylogenetic group
name, and common name information, where available, is
shown in black text in parentheses after the group
name. Each phylogenetic group name is a link that
reveals additional phylogenetic levels (Figure 4and
H-4B.1), allowing the user to navigate onto the
branches of the phylogenetic tree.
In addition to this mode of transversing the
phylogenetic tree, starting at the root and knowing the
pathway to the desired end point, this system has the
facility to jump to specific places in the phylogenetic
tree. The user can enter a partial or complete
scientific or common name in the white text field in
the lower, purple-colored panel of the Selection Frame
(
e.g., "human;" see H-4B.2). Once
the appropriate scientific or common name radio button
is set, different names that satisfy the user-entered
text can be viewed in the Results Frame by checking the
"View" box. Clicking the appropriate name in the
Results Frame will enter that name into the text field;
unchecking the "View" check box and clicking "Submit"
will reveal the phylogenetic branch for this organism
(H-4B.3).
To navigate toward the root of the phylogenetic
tree, click the "Parents" button in the Selection
Frame. This will open a new window with the complete
NCBI phylogeny from the root to the level of the
organism of interest. This window (H-4B.4) also reveals
the phylogenetic level number and common names. Simply
clicking on a node name in this window (
e.g., the "Eutheria" node in
H-4B.4) will reveal this section of the phylogenetic
tree in the Results Frame.
RNA information can be mapped onto the phylogenetic
tree in the Results Frame at any time. In the white
panel in the Selection Frame, the user can choose to
view six RNA types (5S, 16S, and 23S rRNA; group I, II
and other introns) from five cellular locations
(chloroplast, cyanelle, mitochondria, nucleus, and
viral) by checking the boxes to the left of the desired
selections. After clicking the white "Submit" button,
all entries that satisfy the RNA type and cell location
selections are mapped onto the phylogenetic tree in the
Results Frame (H-4B.3). There, the numbers of sequences
and structure diagrams available in our CRW RDBMS are
shown adjacent to each phylogenetic group name at all
levels of the phylogenetic tree and enclosed in
brackets; the format of this information for each
individual RNA type is: [cell location, # sequences/#
structures, cell location, # sequences/# structures,
...]. The RNA types are indicated in different colors
(rRNA: 5S, green; 16S, red; 23S, blue; introns: group
I, black; II, brown; other intron types, magenta) and
the cell locations are abbreviated (N, nucleus; M,
mitochondria; C, chloroplast; Y, cyanelle; V, viral).
These values in brackets link to the Standard RDBMS
results page, as described in the previous section, and
allow the user to view the available sequence and
structure information. The PhyloBrowser page (H-4B.3)
reveals the
"Homo sapiens" phylogenetic group
with the number of sequences and structures available
in our CRW RDBMS for RNA types (
e.g., 16S and group I introns)
that are present in the selected cell locations (
e.g., Chl, Mit, Nuc).
Additional documentation for the use of this page is
available from the PhyloBrowser page. A short
description is displayed in the top-right frame by
placing the mouse over each of the attributes
("Molecule," "Cell Location," "Phylogenetic Levels,"
"Go to Parents," "Query," and "Acknowledgement").
Additional information for each of these attributes is
then displayed in a new window by clicking on either
the attribute link or the additional information link
in the top-right frame (Figure 4and H-4B.1).
4C. RNA Structure Query System
Currently, we are unable to reliably and accurately
predict an RNA structure from its underlying sequence
due in part to the lack of more fundamental RNA
structure rules that relate families of RNA sequences
with specific RNA structural elements. Given this
limitation, we have utilized comparative analysis to
determine that RNA structure that is common to a set of
functionally and structurally equivalent sequences.
This analysis, as mentioned earlier, is very accurate:
nearly 98% of the basepairings in our 16S and 23S rRNA
comparative structure models are present in the
high-resolution crystal structures for the 30S [ 44 ]
and 50S [ 45 ] ribosomal subunits. In the process of
predicting these comparative structure models, we have
determined a large number of 5S, 16S, and 23S rRNA and
group I intron comparative structure models from
sequences that are representative of all types of
structural variations and conservation. Thus, with the
correct rRNA structure models and a large sampling of
structurally diverse structure models, we now want to
decipher more relationships between RNA sequences and
RNA structural elements. Toward this end, we developed
a system for the identification of biases in short
sequences associated with simple structural elements in
our set of comparative structure models. The first set
of examples reveals a sampling of structure-based
sequence biases. Recently, we utilized this system to
identify and quantitate the following biases for
adenosines in the Bacterial 16S and 23S rRNA
covariation-based structure models [ 63 ] : 1)
approximately 2/3 of the adenosines are unpaired; 2)
more than 50% of the 3' ends of loops in the 16S and
23S rRNA have an A; 3) there is a bias for adenosines
to be adjacent to other adenosines (66% of these are at
two unpaired positions, and 15% of these are at
paired/unpaired junctions); and 4) the majority of the
As at the 3' end of loops are adjacent to a paired G.
These results were discerned with this system and are
shown in part in Figure 5and H-4C.
This RNA sequence/structure query system has three
primary fields of input to be selected by the user: the
RNA type, phylogenetic group/cell location, and the
nucleotide/structural element. The options for each of
these fields are listed in Table 5. The system
currently supports four RNA types (5S, 16S, and 23S
rRNAs, and group I introns) and five phylogenetic
groups/cell locations (Bacteria, Archaea, Eucarya
nuclear-encoded, mitochondrial, and chloroplast). Any
combination and number of RNA types and
phylogenetic/cell location groups can be selected,
although at least one RNA type and one
phylogenetic/cell location group must be selected. The
bacterial 16S and 23S rRNAs were selected for the
examples in Figure 5and H-4C. Five nucleotide
categories are searchable: single nucleotides, (two)
adjacent nucleotides, base pairs, three nucleotides,
and four nucleotides. Each category can be searched
against a defined set of structural elements, as
outlined in Table 5. The structural elements for these
nucleotide categories are based on 1) positions that
are paired and unpaired and 2) positions at the center
or 5' and 3' ends of helices and loops.
The sorting function dynamically ranks the
nucleotide patterns. The resulting output reveals, for
any of the selected structural elements, the most
frequent nucleotide pattern, followed by other patterns
in descending order to the least frequent nucleotide
pattern. For the "A Story" example mentioned earlier,
adenosine is the most frequent nucleotide at unpaired
positions (42.64%), followed by G (23.6%), U (21.27%),
and C (12.49%) (Figure 5and H-4C.1). These values are
contained in the orange columns, and reveal the
percentages for each of the nucleotides within each of
the structural elements listed (
i.e., paired, unpaired,
etc. ). This same figure reveals
that 53.5% of the 3' end of loops contain an A. The
unpaired to paired ratio is shown in yellow in Figure
5and H-4C.1; this ratio is greatest for adenosines,
where the value is nearly two (
i.e., there are two unpaired
adenosines for every A that is paired), and lowest for
C, where less than three out of ten cytosines are
unpaired. In contrast with the percentage values in the
orange boxes that reveal the percentage of nucleotides
within each structural element, the percentages in the
green boxes reveal the distribution of nucleotides in
different structural elements for each nucleotide. For
example, 33.76% of the adenosines are paired, while
66.24% are unpaired. In contrast, 77.71% of the C's are
paired and only 22.29% of the C's are unpaired.
The most common adjacent nucleotides in any
structural environment in the Bacterial 16S and 23S
rRNAs are GG (9.86%; H-4C.2), while in loops the most
common dinucleotides are AA (19.2%; H-4C.3), followed
by GA (13.35%), UA (9.821%), AU (6.703%),
etc. The most frequent adjacent
nucleotides at the 3'loop-5'helix junction are AG
(24.99%; H-4C.4), followed by AC (13.28%), GG (8.28%),
etc. For the adjacent AA
sequences, nearly 75% occur in loops, while
approximately 12% of the AA sequences occur in helices,
another 12% occur at the 3'loop-5'helix junction, and
less than 5% occur in 3'helix-5'loop junctions. Thus,
these analyses of single and adjacent nucleotides
reveal several strong biases in the distribution of
nucleotides in different structural environments.
The top section of the output page (Figure 5and
H-4C.1) displays the types of data (RNA molecules and
phylogenetic/cell location groups) that were selected
and analyzed. This section also reveals the number of
structure models that were analyzed; 175 16S and 71 23S
rRNA structure models were analyzed in Figure 5and
H-4C.
A few of the other biases in the distribution of
nucleotide patterns that were determined with this
sequence/structure query system of our comparative
structure models are displayed in Table 6. A more
detailed accounting of this information is available
online.
Auxiliary components of the CRW site
In addition to the sections described above, the CRW
Site also includes online appendices to work published
elsewhere. The "Structure, Motifs, and Folding" section
presently contains three RNA motif projects ("U-Tum" [ 62 ]
, "A Story" [ 63 ] , and "[email protected]" [ 64 ] ) and
two RNA folding projects ("16S rRNA Folding" [ 65 ] and
"23S rRNA Folding" [ 66 ] ). In the "Phylogenetic Structure
Analysis" section, additional information for three
publications is available: "Mollusk Mitochondria" [ 67 ] ,
"Polytoma Leucoplasts" [ 68 ] , and
"Algal Introns" [ 69 ] .
Conclusions
Nearly 10 years ago, our initial goals for our RNA web
page was to disseminate some of the comparative information
we collected and analyzed for our prediction of 16S and 23S
rRNA structure with comparative analysis. With dramatic
increases in the number of ribosomal RNA sequences, we
developed a relational database system to organize basic
information about each sequence and structure entry to
maintain an inventory of our collection, and to retrieve
any one or set of entries that satisfy the conditions of
the search. In parallel, with the significant advancements
in computational and networking hardware and software, our
need for more detailed and quantitative comparative
information for each RNA molecule under study, and our
interest in studying more RNA molecules beyond 16S and 23S
rRNA, we have greatly expanded our web site, and named it
the "Comparative RNA Web" (CRW) Site.
The major types of information available for each RNA
molecule are:
1) the current comparative RNA structure model;
2) nucleotide and base pair frequency tables for all
positions in the reference structure;
3) secondary structure conservation diagrams that reveal
the extent of conservation in the RNA sequence and
structure;
4) representative secondary structure diagrams for
organisms from phylogenetic groups that span the
phylogenetic tree and reveal the major forms of structural
variation;
5) a semi-complete/partial collection of publicly
available sequences that are 90% or more complete; and
6) sequence alignments.
At this time, we maintain the most current comparative
sequence and structure information about the 16S and 23S
rRNA. The other RNA molecules we maintain (5S rRNA, tRNA,
and group I and II introns) are not as advanced at the time
of this writing.
Our future aims for the CRW Site are to: 1) maintain a
complete collection of sequences in our database management
system for each of the RNAs under study; 2) once or twice a
year, release new sequence alignments that contain A)
improvements (if necessary) in the positioning of the
sequences that are associated with similar structural
elements, and B) increases in the number of aligned
sequences; 3) generate more secondary structure diagrams
for sequences that span the phylogenetic tree and reveal
all forms of structural variation; 4) generate more
secondary structure conservation diagrams and nucleotide
and base pair frequency tables for more phylogenetic groups
(
e.g. Fungi: Basidiomycota,
Ascomycota, and Zygomycota); 5) update the structure models
when warranted by the analysis; 6) update current
nucleotide and base pair frequency tables when the
alignments they are derived from have been updated, and
generate more frequency tables for more phylogenetic groups
(see "4)" above); 7) add new types of comparative RNA
sequence/structure information and new modes of presenting
the data; and 8) analyze more types of RNA molecules from a
comparative perspective, and present this data in the same
formats utilized for the RNA molecules currently
supported.
Materials and Methods
Sequence collection
The majority of the sequence alignments presented at
the CRW Site were assembled in the Gutell laboratory. The
alignments that were based on another laboratory's
initial effort and enlarged and refined for the CRW
project are: 1) the prokaryotic (Archaea and Bacteria)
alignments for 16S rRNA [ 85 ] ; 2) the 5S rRNA
alignments [ 55 ] ; and 3) the tRNA alignments [ 81 ] .
The group I and II intron alignments were originally
based upon sequences collected by Michel [ 32 34 ] .
New rRNA and intron sequences were found by searching
the nucleic acid sequence database at GenBank using the
NCBI Entrez system http://www.ncbi.nlm.nih.gov/Entrez/at
least once per week with appropriate search criteria (
e.g., "rrna" [Feature key] and
"intron" [Feature key] to find introns that occur in
rRNA). While the majority of the RNA sequences of
importance to this database are available online at
GenBank, a few sequences are only available in the
literature (
e.g., the
Urospora penicilliformis intron [
86 ] ) or in a thesis; these sequences were manually
entered into the appropriate sequence alignment. A few
sequences were found in GenBank with the sequence
similarity searching program BLAST [ 87 ] . At this time,
we are only trying to identify all sequences that are
more than 90% complete since all sequences that are less
than 90% complete are not currently retrieved with the
CRW RDBMS.
Deviations in GenBank entries
The majority of GenBank entries contain accurate
annotations of the RNAs. However, some GenBank entries
deviate from this norm in a variety of ways. In some
entries, the presence of the rRNA was not annotated and
the rRNA was found by searching for short sequences that
are characteristic of that rRNA (a few examples).
Sometimes, intron sequences are not annotated and were
discovered during the alignment of the corresponding rRNA
exons (
e.g., the unannotated intron in the
uncultured archaeon SAGMA-B 16S rRNA (AB050206) and many
Fungi, including AF401965 [ 88 ] ). Other GenBank entries
contain incorrect annotations for the RNAs; the
boundaries may be misidentified by a small or large
number of nucleotides.
RNA sequence alignment and classification of intron
sequences
Alignment and determination of intron-exon
boundaries
The sequence alignments used for this analysis are
maintained by us at the University of Texas; these
alignments, containing all publicly available sequences
used in the analysis, are or will be available from the
CRW Site http://www.rna.icmb.utexas.edu(Table 2). rRNA,
Type 1 tRNA, and intron sequences were manually aligned
to maximize sequence and structural identity using the
alignment editor AE2 (T. Macke, Scripps Clinic, San
Diego, CA). The rRNA alignments are sorted by phylogeny
and cell location, the intron alignments are sorted by
subgroup, exon, insertion point (for rRNA introns), and
phylogeny, and the tRNA alignments are sorted by
aminoacyl type and phylogeny. Alignment of the rRNA
exons (when available) between closely-related
sequences provided an independent evaluation of the
intron-exon borders for each intron-containing rRNA
sequence; the large number of rRNA sequences in our
collection and the high level of sequence conservation
at intron insertion points provide great confidence in
this evaluation.
Classification of introns
Group I and II intron sequences were classified into
one of the structural subgroups defined by Michel [ 32
34 ] or the more recently determined subgroup IE [ 82 ]
based upon sequence and structural homology to
previously-aligned sequences. Uncertainties in these
assignments come from two main sources. First, some
introns are referred to in rRNA GenBank entries without
the intron sequence being provided; in these cases, we
represent the intron as having length "NSEQ" (No
SEQuence information) and accept the authors' major
intron classification (
e.g., group I or group II) but
not the specific intron type (
e.g., if an author classified an
intron as IA1 and did not publish the sequence, our
system designates its type as "I"). In the second case,
we do have sequence information but cannot fully
classify the intron with confidence; here, we provide
the most plausible classification. The classifications
"I" and "II," respectively, are group I and II introns
of undefined subtype. An intron described as "IB' has
the characteristic features of the IB subgroup but
cannot be subclassified as IB1, IB2, IB3, or IB4. Those
introns that do not belong to either group I or group
II are generally classified as "Unknown" in the "RNA
Class" field (see Section 4A and Table 5); included in
this category are the Archaeal and spliceosomal
introns. At present, the Archaeal and spliceosomal
introns are identified with the phrases "Archaeal" and
"spliceosomal," respectively, in the Comment field of
the RDBMS; a standard designation for these introns
will be added to a future version of the system.
Although the introns in our collection have been
judiciously placed into one of the intron subgroups and
are roughly correct, these intron placements will be
reanalyzed to assure the accurate assignment of
subgroups.
Identification of unannotated or misannotated
introns, with examples
Some examples of introns that were identified or
clarified by the alignment process are: 1)
Aureoumbra lagunensis (U40258;
the intron was annotated as an insertion); 2)
Exophiala dermatitidis (X78481;
the intron was not annotated); and 3)
Chara sp. Qiu 96222 (AF191800;
the intron annotations were shifted approximately 15
positions toward the 5' end of the rRNA sequence).
About TBD and NSEQ
Information that could not be determined either from
the GenBank entries or by using these methods is
represented in the RDBMS system as TBD (To Be
Determined). When a sequence is known but not available
(for example, when an intron is inferred from a rRNA
GenBank entry), the sequence length and percent
completeness are instead represented as NSEQ (No
SEQuence), to show that the sequence itself is not
available.
Database System
Contents of the RDBMS (general and
intron-specific)
The relational database management system (RDBMS)
available from the Comparative RNA Web Site
http://www.rna.icmb.utexas.edudescribed in this work
utilizes the MySQL engine http://www.mysql.com/. The
system contains vital statistics for each sequence
(Table 4). The primary fields are: 1) organism name; 2)
complete phylogeny; 3) cell location; 4) RNA type
(general category;
e.g., rRNA or intron); 5) RNA
class (more detailed identification;
e.g., 16S or IC1); 6) GenBank
Accession Number (linked to GenBank); and 7) secondary
structure diagrams for selected sequences.
Intron-specific data stored in the system are the exon,
intron number (index for multiple introns from a single
exon), intron position (for rRNA introns only: the
E. coli (GenBank Accession Number
J01695) equivalent position number immediately before
the intron), and open reading frame presence. Note that
only sequences that are at least 90% complete are made
available through this system. The majority of this
data is manually entered into the database system; one
exception is the complete NCBI phylogeny database [ 60
61 ] , which is automatically downloaded and
incorporated into this system daily so that all RDBMS
entries appear using the current NCBI scientific name
for a given organism. Changes to the RDBMS phylogeny
data are identified automatically during the
incorporation process and then updated manually. Any
changes made to the data become available to the public
on the next day.
Secondary Structure and Conservation
Diagrams
Secondary structure and conservation diagrams were
developed entirely or in part with the interactive
graphics program XRNA (Weiser & Noller, University
of California, Santa Cruz). The PostScript files output
by XRNA were converted into PDF using ghostscript
(version 7.00;
http://www.cs.wisc.edu/~ghost/index.htm).
Computer details
Hardware and software used
The Comparative RNA Web Site
http://www.rna.icmb.utexas.eduis hosted on a Sun
Microsystems Enterprise 250 dual-processor server.
Apache web server version 1.3.20, from the Apache
Software Foundation http://www.apache.org/, provides
the site's connectivity interface. The MySQL database
(version 3.23.29; http://www.mysql.com/) provides the
RDBMS functions. Web site statistics are collected
using webalizer (version 2.01;
http://www.mrunix.net/webalizer/).
Authentication system
The Comparative RNA Web Site has instituted an
authorization system for its users. Information is
collected to assist in web server administration and
error tracking. On their initial visits, users will
select a username, provide a current email address (for
verificiation purposes), and review the terms and
conditions for use of the CRW Site. An email will be
sent to the provided email address containing a
validation URL for that account. At this URL, the user
may provide additional information; the system will
then email an initial password to the user at the
selected email account. The user then has the two
pieces of information (username and password) necessary
to log in and use the CRW Site. Once logged in, the
user may change the password and update the user
information at any time.
URL rewriting
We strongly encourage all users to access the
Comparative RNA Web Site
http://www.rna.icmb.utexas.eduusing its main address,
http://www.rna.icmb.utexas.edu/, rather than through
specific URLs. As the site grows, specific pages may be
moved, changed, or deleted. As well, use of more
specific URLs may not include the navigation system for
the site, providing the user with a suboptimal
operating experience of the entire site. Therefore, the
system is configured to route an initial request for a
more specific URL to an introductory page, which will
offer users access to the main page and a selection of
specific URLs.
List of abbreviations
CRW = Comparative RNA Web
NCBI = National Center for Biotechnology
Information.
nt = nucleotide
RDBMS = Relational Database Management System.
URL = Uniform Resource Locator
