Gutell 080.bmc.bioinformatics.2002.3.2

of 31
(page number not for citation purposes)
BMC Bioinformatics
BMC Bioinformatics2002, 3
Research article
The Comparative RNA Web (CRW) Site: an online database of
comparative sequence and structure information for ribosomal,
intron, and other RNAs
Jamie J Cannone1, Sankar Subramanian1,2, Murray N Schnare3,
James R Collett1, Lisa M D'Souza1, Yushi Du1, Brian Feng1, Nan Lin1,
Lakshmi V Madabusi1,4, Kirsten M Müller1,5, Nupur Pande1, Zhidi Shang1,
Nan Yu1 and Robin R Gutell*1
Address: 1Institute for Cellular and Molecular Biology, Section of Integrative Biology, University of Texas at Austin, 2500 Speedway, Austin, TX
78712-1095, USA, 2Department of Biology, Arizona State University, Tempe, AZ 85287-1501, USA, 3Department of Biochemistry and Molecular
Biology, Dalhousie University, Halifax, Nova Scotia B3H 4H7, Canada, 4Ambion, Inc., Austin, TX 78744-1832, USA and 5Department of Biology,
University of Waterloo, Waterloo, Ontario N2L 3G1, Canada
E-mail: Jamie J Cannone - cannone@mail.utexas.edu; Sankar Subramanian - sankar@asu.edu;
Murray N Schnare - mschnare@rsu.biochem.dal.ca; James R Collett - colletj@ccwf.cc.utexas.edu; Lisa M D'Souza - lisadsouza@mail.utexas.edu;
Yushi Du - ysdu@cs.utexas.edu; Brian Feng - bfeng@mail.utexas.edu; Nan Lin - nanlinemail@yahoo.com;
Lakshmi V Madabusi - lmadabusi@ambion.com; Kirsten M Müller - kmmuller@sciborg.uwaterloo.ca; Nupur Pande - nupur@mail.utexas.edu;
Zhidi Shang - shangzd2001@yahoo.com; Nan Yu - nanyu@mail.utexas.edu; Robin R Gutell* - robin.gutell@mail.utexas.edu
*Corresponding author
Abstract
Background: Comparative analysis of RNA sequences is the basis for the detailed and accurate
predictions of RNA structure and the determination of phylogenetic relationships for organisms
that span the entire phylogenetic tree. Underlying these accomplishments are very large, well-
organized, and processed collections of RNA sequences. This data, starting with the sequences
organized into a database management system and aligned to reveal their higher-order structure,
and patterns of conservation and variation for organisms that span the phylogenetic tree, has been
collected and analyzed. This type of information can be fundamental for and have an influence on
the study of phylogenetic relationships, RNA structure, and the melding of these two fields.
Results: We have prepared a large web site that disseminates our comparative sequence and
structure models and data. The four major types of comparative information and systems available
for the three ribosomal RNAs (5S, 16S, and 23S rRNA), transfer RNA (tRNA), and two of the
catalytic intron RNAs (group I and group II) are: (1) Current Comparative Structure Models; (2)
Nucleotide Frequency and Conservation Information; (3) Sequence and Structure Data; and (4)
Data Access Systems.
Conclusions: This online RNA sequence and structure information, the result of extensive
analysis, interpretation, data collection, and computer program and web development, is accessible
at our Comparative RNA Web (CRW) Site [http://www.rna.icmb.utexas.edu] . In the future, more
data and information will be added to these existing categories, new categories will be developed,
and additional RNAs will be studied and presented at the CRW Site.
Published: 17 January 2002
BMC Bioinformatics 2002, 3:2
Received: 7 December 2001
Accepted: 17 January 2002
This article is available from: http://www.biomedcentral.com/1471-2105/3/2
© 2002 Cannone et al; licensee BioMed Central Ltd. Verbatim copying and redistribution of this article are permitted in any medium for any purpose,
provided this notice is preserved along with the article's original URL.

BMC Bioinformatics 2002, 3 http://www.biomedcentral.com/1471-2105/3/2
Page 2 of 31
Background
In the 1830's, Charles Darwin's investigation of the Gala-
pagos finches led to an appreciation of the structural char-
acteristics 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 the-
ory 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 hard-
ware and software, affords us the opportunity to deter-
mine 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 evolu-
tion from a molecular sequence and structure perspective.
Their two primary objectives were to determine phyloge-
netic 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 relation-
ships for all organisms are encoded in the organism's rR-
NA, 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 signifi-
cant findings was the discovery of the third kingdom of
life, the Archaebacteria (later renamed Archaea) [2–4].
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 sequenc-
ing of more than 10,000 16S and 16S-like rRNA and
1,000 23S and 23S-like rRNA genes, from laboratories try-
ing to resolve the phylogenetic relationships for organ-
isms that occupy different sections of the big phylogenetic
tree.
The prediction of tRNA structure with a comparative per-
spective in the 1960's [5–9] and subsequent validation
with tRNA crystal structures [10,11] established the foun-
dation for Woese and Fox in the 1970's to begin predict-
ing 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 simi-
lar to the correct secondary structure for every other 5S
rRNA sequence [12,13].
While the first complete 16S rRNA sequence was deter-
mined for E. coli in 1978 [14], the first covariation-based
structure models were not predicted until more 16S rRNA
sequences were determined [15–17]. The first 23S rRNA
sequence was determined for E. coli in 1980 [18]; the first
covariation-based structure models were predicted the fol-
lowing year, once a few more complete 23S rRNA se-
quences were determined [19–21]. 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 re-
solve different types and extents of covariation (see be-
low). 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 nu-
merous revisions [23–28]. Today, with a significantly larg-
er number of sequences and more advanced covariation
algorithms, we search for all positional covariations, re-
gardless 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 covari-
ation-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 sec-
ondary structure helices, there were many novel base-pair-
ing 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 predic-
tion of structure and the identification of structural motifs
[29].
Beyond the comparative structure analysis of the three ri-
bosomal RNAs and transfer RNA, several other RNAs have
been studied with this perspective. These include the
group I [30–33] and II [34,35] introns, RNase P [36–38],
telomerase RNA [39,40], tmRNA [41], U RNA [42], and
the SRP RNA [43]. The comparative sequence analysis par-
adigm 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 im-
plementation of this method, including the algorithms,

Page 3 of 31
the sequence alignments, and the large collection of com-
parative structure models with different structural varia-
tions 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 analy-
sis 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, ana-
lyzing, 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 [48–52] and the group I introns [33]. In parallel,
two other groups have been providing various forms of ri-
bosomal 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 signifi-
cant 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 soft-
ware, we have established a new "Comparative RNA Web"
(CRW) Site [http://www.rna.icmb.utexas.edu/] . This re-
source has been available to the public since January
2000.
Results and Discussion
The primary objectives and accomplishments for our
Comparative RNA Web (CRW) Site are:
I. To study the following RNA molecules from a compar-
ative perspective:
A. Primary importance: 16S and 23S rRNA.
B. Secondary importance: 5S rRNA, tRNA, group I and II
introns.
II. To provide the following comparative information for
each of these RNA molecules:
A. The newest comparative structure models for the pri-
mary RNA types.
B. Nucleotide frequency tables for all individual posi-
tions, base pairs and base triples in the comparative struc-
ture models. This nucleotide frequency information is
also mapped onto the complete NCBI phylogenetic tree
[60,61], revealing the type and extent of sequence and
base pair conservation and variation at each position in
the 16S and 23 S rRNAs at each node in the phylogenetic
tree.
C. A phylogenetic and structurally diverse set of secondary
structure models (with diagrams and lists of positions that
are base-paired) for each of the RNA types in this collec-
tion.
D. Secondary structure diagrams revealing the extent of se-
quence and structure conservation for different phyloge-
netic groups at different levels in the phylogenetic tree.
E. Basic information (organism name, RNA type, length,
etc.) and NCBI GenBank [60] entries for each RNA se-
quence that is analyzed within the CRW Site.
F. Sequence alignments created and maintained for com-
parative structure analysis.
III. To catalog portions of this information in our relation-
al database management system (RDBMS) and to dynam-
ically retrieve it from our summary pages, full relational
search, and phylogenetic tree-based search systems.
IV. To present additional pages that:
A. Reveal the evolution of the 16S and 23S rRNA structure
models.
B. Describe the comparative and covariation analysis tech-
niques that we have utilized within the CRW Site.
C. Formally define each of the primary RNA structure ele-
ments.
D. Contain figures and data tables for our own publica-
tions detailing RNA structural motifs from a comparative
perspective:
1. "Predicting U-turns in the ribosomal RNAs with com-
parative sequence analysis" [62].
2. "A Story: unpaired adenosines in the ribosomal RNAs"
[63].
3. "AA.AG@helix.ends: AA and AG base-pairs at the ends
of 16S and 23S rRNA helices" [64].
E. Contain figures and data tables for our own publica-
tions addressing RNA folding:
1. "A comparison of thermodynamic foldings with com-
paratively derived structures of 16S and 16S-like
rRNAs"[65].

Page 4 of 31
2. "An Analysis of Large rRNA Sequences Folded by a
Thermodynamic Method" [66].
F. Contain figures and data tables for our own publica-
tions that analyze RNA structure from a phylogenetic per-
spective:
1. "Phylogenetic Analysis of Molluscan Mitochondrial
LSU rDNA Sequences and Secondary Structures" [67].
2. "Accelerated Evolution of Functional Plastid rRNA and
Elongation Factor Genes Due to Reduced Protein Synthet-
ic Load After the Loss of Photosynthesis in the Chloro-
phyte Alga Polytoma" [68].
3. "Group I Intron Lateral Transfer Between Red and
Brown Algal Ribosomal RNA" [69].
The contents of our Comparative RNA Web (CRW) Site
are outlined on its main page [http://www.rna.icmb.utex-
as.edu/] (Figure 1). The detailed explanations of the data
and their presentations in the first four sections of this site
(1. Comparative Structure Models; 2. Nucleotide Frequen-
cy and Conservation Information; 3. Sequence and Struc-
ture Data; and 4. Data Access Systems) are presented here.
To fully appreciate this description of the CRW Site, we
encourage users to evaluate the pages at this web site while
reading this manuscript; while a few of the pages and links
at the CRW Site are shown as figures here, the reader is
routinely referred to the actual web pages and the corre-
sponding highlights on the "Table of Contents."
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 [5–9],
and the group I [32] and group II [34] introns, as deter-
mined by others. This collection of RNA structure models
was predicted with covariation analysis, as described at
the CRW Site Methods Section [http://www.rna.ic-
mb.utexas.edu/METHODS/] and in several publications
(see below).
Briefly, covariation analysis, a specific application of com-
parative analysis (as mentioned earlier), searches for heli-
ces 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 se-
quences that had similar patterns of variation. This latter
implementation of the covariation analysis helped us re-
fine the secondary and tertiary structure models by elimi-
nating 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 [19,70–72]. Our newest cov-
ariation analysis methods associate color-coded confi-
dence ratings with each proposed base pair (see reference
structure diagrams and Section 2A, "Nucleotide Frequen-
cy 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
[5–9,71–74] 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
Figure 1
Introductory view of the CRW Site. The top frame divides
the site into eight sections; the first four sections are the pri-
mary focus of this manuscript. The bottom frame contains
the CRW Site's Table of Contents. Color-coding is used con-
sistently throughout the CRW Site to help orient users.

Page 5 of 31
pairs predicted with covariation analysis are in these crys-
tal 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 terti-
ary 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 struc-
tural 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 Organ-
isms" page, our primary focus has been on the 16S and
23S rRNAs. Thus, some of our subsequent analysis and in-
terpretation will emphasize only these two RNAs.
Each RNA structure model presented here is based upon a
single reference sequence, chosen as the most representa-
tive 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 Struc-
ture Models for Reference Organisms" page [http://
www.rna.icmb.utexas.edu/CSI/2STR/] contains six or sev-
en different diagrams for that molecule: Nucleotide, Ten-
tative, Helix Numbering, Schematic, Histogram, Circular,
and Matrix of All Possible Helices.
Nucleotide: The standard format for the secondary struc-
ture 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 pre-
dicted 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 mod-
els are shown in Figures 2A, 2B, and 2C. Note that the ma-
jority 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 Infor-
mation" and in Table 2. These sequences have been
aligned and analyzed with several covariation algorithms,
as described in more detail in the "Predicting RNA Struc-
ture 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 stand-
Figure 2
The most recent (November 1999) versions of the rRNA
comparative structure models (see text for additional
details). A. E. coli 23S rRNA, 5' half. B. E. coli 23S rRNA, 3'
half. C. E. coli 16S rRNA. D. The "histogram" format for the
E. coli 16S rRNA.
Secondary Structure: small subunit ribosomal RNA
Escherichia coli
November 1999 (cosmetic changes July 2001)
(J01695)
10
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
5’
3’
I
II
III
m
2m
5
m7
m
2
mm
4
m5
m2
m
6
2
m6
2
m3
G[ ]
Symbols Used In This Diagram:
G A
- Canonical base pair (A-U, G-C)
- G-A base pair
- G-U base pair
G C
G U
U U - Non-canonical base pair
Citation and related information available at http://www.rna.icmb.utexas.edu
Every 10th nucleotide is marked with a tick mark,
and every 50th nucleotide is numbered.
Tertiary interactions with strong comparative data are connected by
solid lines.
1.cellular organisms 2.Bacteria 3.Proteobacteria
4.gamma subdivision
5. Enterobacteriaceae and related symbionts
6. Enterobacteriaceae 7. Escherichia
A
A
A
U
U
G
A
A
G A G U U
U G
A
UCAUGGCUCAG
A
U
U
GA
A
C
G
C
U
GG
C
G
G
C
A
G
G
C
C
UA
AC
A
C A
U
G
C
A
A
G U C
G A
A C G G U
A A
C A G G A A G A A G C
U
U
GCUUCUUU
G
CUGAC
G
AGUGGC
G
G
A
CGG
G
U
G
A
G
U
A
A
UG
U
C
U
G
G
G
A
A
A
C
U
G
C
C
U
G
A
U
G
G
A G G G G
GA U A
A C U A C U G G
A
A
ACGGUAGC
U
AAU
A
CCGC
A
U
A
A
C
G
U
C
G
CA
A
G
A
C
C
A
A
A
GAGGGG
GA
CCU
U
C
G G G C C U C U U G
C
C
A
U
C
G
G
A
U
G
U
G
C
C
C
A
G
A
UG
G
G
A
UU
A
G
C
U
A
GU
A
G
G
U
G
G
G
G
UA
A
C
G
G C
U
C
A
C
C
U
A
G
G
C
G
A
C G A
U
C
C
C
U
A
GCUG
GUCU
G
A
G A
GGA U
G A
C
C A GC C
A
C
A
CUGGAA
CUG
A
G
A
CA
C G
G U C C A G
A
C
U
C
C
U
A
C G
G
G
A
G
G C A G
C
A
G
U
G
G
G
G
A
A
U
AU
U
GCA
CAA
UGGGCG
C
A
A G C C U G A U G C A GC
C
A U
G
C
C
G
CGUGUAU
G
AAGA
A
GGCCU
U
C
G G G U U
G
U A A
A
G U A C
U
U
U
C
A
G
C
G
G
GG
A
G
GAA
G
G
G
A
G
U
A
A
A
GU
U
A
A U A
C
C
U
U
U
G
C
U
CA U
U
G
A
C G U
U
A
C
C
C
G
C
A
G
A
A
G
A
AG
C
A
C
CGGC
UA A C
U
C
C
G
ψ
G
C
C
A
G
C
A
G C C
G
C G
G
U
A
A
U
AC
G
G
A
G
G
G
U
G
C
A
A
G
C
G
U
U
A
A
U
C
G
G
A
A
U
U
A
C
U
G G
G
C
GU
A
A
A
G
C
G
C
A
CG
CA
G
G
C
GGUUUGUU
A
AGUCAGAUGUG
A
AA
U
CCCCGGGCU
C
A A C C U G G G A
A C
U G C A U C U G A
U A
C U G G C A A G C
U
U
G
A
G
U
C
U
C
G
U
A
G
A
G
G
G
G
G
G
U
AGAAUUCCAGGU
GUA
GCGGU
G
A
A A U G C
G
U
A G
A
G
A U C U G G A G G A A U
A
C C
G
G
U G
G C G
A
A
GGCG
G
C
C
C
C
C
U
G
G
A
C
G
A
A
G
A
C
U
G
A
C
G
C
U
C
A
G
G
U
G
CG
A
A
A
G
C
G
U
G
GG
G
A G
C
A
A
A
C
A
G
G
A
U
U
A G A
U
A
C
C
C
U
G
G
U
A
G
U
C
C
A
C
G
C C G U
A
A
A
C
G
AU
G U C G A C U U G
G
A
G
G
U
U
G
U
G
C
C
C U U
G
A
G
G
C
G
U
G
G
C
U
U
C
CG
G
A
G
C
U
A
AC
G
CGU
U
A
A
GUCGAC
C
G
C
C
U
G G G
G
A G U A
C
G G C C G
C
A
AGGUU
AAAA
CUC
A
A A
U G A A U U G A C G
G
G G G C C C G
C
A C A A GC
GG
U
G
G
A
G
C
A
U
G
U
G
G
UU
UAAU
U
C
G
A
U
GC
A
A
C
G C
G
A
A
G
A
A
C C U U
A
C
C
U
G
G
U
CU
U
GA
C
A
U
C
C
A
C
G
GAAGUUUUCAG
A
G
A U G A G A A U G
U
G
C
C
U
U C
G
G
G
A
A
C
C
G
U
GA
G
A
C A
G
G
U
G
C
U
GC
A U
G
G
C
U
G
U
C
G
U
C
A
GCUCGUG
U
U
G
UG
A
A
A
U
G
U
U
G
G
G
U
U
A A
G
U
C
C
C
G C
A
A C G A G C
G
C A A
C
C C U U A U C C U U U G U U G C C
A G
C G G U
C
C
GGCCGGG
AACU
CAAAGGA
G
A
C
U
G
C
C
A
G
U
G
AUA
A
A
C
U
G
G
A
G
G
A
A
G
G
UGGGGA
U
G
A
C
G
U
C
A
A
G
U C
A
UC
A
U
G
G
C
C
C
U
U
A
CG
A
C
C
A
G
G
G
C
U
A
C
A
C
A
C
G
U
G
C
U
A
C A A
U G
G
C
G
C
A
U
A
C
A A A G
A
G
A
A G
C
G
A C C
U
C
G C
G
A
G
A
G
C
AA
G
C
G
G
AC
C
U
C
A
U
AAAG
U
G
C
G
U
C
G
U
A
G
U
C
C
G
G
A
U
U
G
G
A
G
U
C
U
G
C
AAC
U
C
G
A
C
U
C
C
A
U
G
A
A
GU
C
G
G
A
A
U
C
G
C
U
A
G
U
A
A
U
C
G
U
G
G
A
U
C
A
GAA
U
G
C
C
A
C
G
G
UG
A
A
U
A
C
GU
U
C
C
CGGGCCUUGU
A
CA
C
A
C
C
G
C
C
C
G
U
C
A
C
A
C
C
A
U
G
G
G
A
G
U
G
G
G
U
U
G
C
A
A
A
A
G
A
A
G
U
A
G
G
U
A
G
C
U
U
A
A
C
C
U
U C
G
G
G
A
G
G
G
C
G
C
U
U
A
C
C
A
C
U
U
U
G
U
G
A
U
U
C
A
U
G
A
C
U
G
G
G
G
U
GA
AG
U
C
GU
A
A
C
A A
G
G
U A A C C G U A G G
G
G
A
A
CCUGCGGUUG
G
A
U
C
A
C
C
U
C
C
U
U
A
Secondary Structure: large subunit ribosomal RNA - 3’ half
Escherichia coli
(J01695)
1.cellular organisms 2.Bacteria 3. Proteobacteria
4.gamma subdivision
5.Enterobacteriaceae and related symbionts
6.Enterobacteriaceae 7.Escherichia
November 1999 (cosmetic changes July 2001)
G C - Canonical base pair (A-U, G-C)
G U - G-U base pair
G A - G-A base pair
Every 10th nucleotide is marked
with a tick mark, and every 50th
nucleotide is numbered.
Tertiary interactions with strong
comparative data are connected by
solid lines.
IV
V
VI
5’
3’
1650
1700
1750
1800
1850
1900
1950
2000
2050
2100
2150
2200
2250
2300
2350
2400
2450
2500
2550
2600
2650
2700
2750
2800
2850
2900
5’ half
m2
m
3
m
5
m
6m
7
m
m
m
2
(1269-1270)
(413-416)
(1262-1263)
(746)
(531)
5
m
m
-[m 2G]
G
G
U
U
A
A
G
C
U U
G
A
GA
G
A
A C
U
C
G
G
G
U
G
A
A
G
GAACUAGGCAAAAUGGUGCC
GUA
ACU
U
C
G G G
A G A A
G G C A C
G
C
U
G
A
U
A
U
GU
A
GG
U
G
A
GG
U
C
C
C
U
C G
C
G
G
A
U
G
G
A
G
C
U
G
A
A
A
U
C
A
G
U C
GA A
G A U A C C A G C
U
G
G
C
U
G
C
A
A
C
UGU
UUA
U
U
A
A A A
A C A
C
A
G
C
A
C
U
G
U
G
C
A
A
A
C
A
C
G
A A
A
G
U
G
G
A
C
GU
AU
A
C
G
G
U
G
U
G
A
C G C C
U
G
C
CC
G G
U
G
C
C
G
GA
A G
G
U
U
A
A
U
U
G
A
U
G
G
G
G
U
U
A
G
C
G
C A
A
G
C
G
A
A
G
C
U
C
U
U
G
A
U
C
G
A
A
G
C
C
C
C
G
G
U A
AA
C
G
G
C G
G
C
C
G
ψ
A
AC
ψ
A
ψ
A
A
C
G
G
U
C C
U A
A
G
G
U
A
G
C
G
A
A
A
U
U
CCUUG
U
C
G
G
G
U
AAG
U
U
C
C
G
A
CC
U
G
C
A
C
G
A
A
U
GGCG
U
A
AU
GA
U
G
G
C
C
A
G
G
C
U
G
U
C
U
C
C
A
C
C
C
G
A
G
A
C
U
C
A GU G A A A
U
U
G
A
A
C
U
C GC U G
U
G A
A
G
A
UGCAGUG
U
A
C C C G C G G C
A
A G A C G G
A
A
A
G
A C
C
C
C
GU
G
A
A
C
C
U
U
U
A
C
U
A
U
A
G
C
U
U
G
A
C
A
C
U
G
A
A
C
A
U
U
G
A
G
C
C
U
U
G
A
U
G
U
G
U
A
G
G A U
A
G G U G G
G
A G
G
CU
U
U
G
A
A G
U
G
U
G
G
A
C
G
C C
A
G
U
C
U
G
C
A
U
G
G
A
G
C
C
G
A
C
C
U
U
GAAAU
A
CCACCC
U
U
U
A
A
U
G
U
U
U
G
A
U
G
U
U
C U A A C G U
U
G A C C C G U A
A
UCCGGGUUGCG
G
ACAGU
G
U
C
U
G
G
U
G
GG
U
A
G
U
U U G
A
C
U
G
G G G
C
G
G
U
C U
C
C
U
C
C
U
A
A
A
G A G
U
A
A
C
G
G
A
G
G
A G C A C
G
A
A
G
G
U
U
G
G
C
U
A
A
U
C
C
U
G
G
U
C
G G A
C
A
U
C
A
G
G
A G
G
U
U
A G
U
GC A
A
U
G
G
C
A
UA
AG
C
C
A
G
C
U
U
G
A
C U G C G A G C G U G
A
C
GGCGCGAGCAG
G
U
G
C
G
AA
A
G
C
A
G
GU
C
A
U
A
GU
G
A
U
CC
G
G
U
G
G
U UC
U
G
A
A
UG
G
A
A
G
G
G
C
C
A
U
C
GC
U
C
A
ACG
G
A
U
A
AA
A
G
G
U A
CU
C
C
G
G
G
G A D
A
A
C
A
G
G C ψ
G
A U A C C G C C
C A A
G A
G U
U
C
A
UA
UC
GAC
GGCGGUG
UU
UGGC
A
C
C
U
C
G
A
ψGUC
G
G
C
U
C
A
U
C
A
C
A U C C U G G G G C U G A
A
G
UAGGUCCC
AA
GGGU
A
U
G
G
C
U
G
U
U
C
G
C
C
A
UU
U
A
A
A G
U
G
G
UA
C
GC
GA
G
C
ψ
G
GGUUU
A
G
A
A
C
G
U
C
GU
G
A
G
A
C
A G
U
ψ
C
G
G
U
C
CC
UA
UCUGCCGUGGG
C
G
C
U
G
G
A
G
A
A
C
U G
A
G
G
G
GGG
C
U
G
C
U
C
C
U
A G
U
A C
G A
G
A
G
GA
C
CG
G
A
G
U
G
G
A
C
G
C
A
UC A
C
U
G
GU G
U
U
C
G
G
G
U
U
G
U
C
A
U
G
C
CA
A
U
G
G
C
AC
U
G
C
C
C
GGU
A
G
C
U
AA
A
U
G
C
G
G
AAG
A
G
A
U
AAG
U
G
C
U
G
A
AAG
C
A
U
C
U A A
G
C
A
C
G
A
A A C
U
U
G
C
C
C
C
GAG
A
U
G
A
G
U
U
C
U
C
C
C
U
G
A
C
C
C
U
UU
A
A
G
G
G
U
CCUGAAG
G
A
A C G U U G
A A
G
A
C
GA
CGACG
U
U
GAU
A
G
G
C
C
G
G
G
U
G
U
G
U A
AG
C
G
C
A
G
CG
A
U
G
C
G
U
U
G
A
G
C
U
A
A
C
C
G
G
U
A C
U
A
A
U
G
A
A
C
CGUGA
GG
C
U
U
A
A
C
C
U
U
Secondary Structure: large subunit ribosomal RNA - 5’ half
Escherichia coli
(J01695)
1.cellular organisms 2.Bacteria
3.Proteobacteria
4.gamma subdivision
5.Enterobacteriaceae and
related symbionts
6.Enterobacteriaceae
7.Escherichia
November 1999
(cosmetic changes July 2001)
G A
- Canonical base pair (A-U, G-C)
- G-A base pair
- G-U base pair
G C
G U
Every 10th nucleotide is marked with a tick
mark, and every 50th nucleotide is numbered.
Tertiary interactions with strong comparative
data are connected by solid lines.
I
II
III
50
100
150
200
250
300
350
400
450
500
550
600
650
700
750
800
850
900
950
1000
1050
1100
1150
1200
1250
1300
1350
1400
1450
1500
1550
1600
1640
2900
5’ 3’
3’ half
m1
m
5
m
6
(2407-2410)
(2010-2011)
(2018)
(2057/2611 BP)
(2016-2017)
G
G
U
U
A
A
G
C
G
A
C
UAAG
C
G
U
A
C
A
C
G
G
U
G
G
A
U
G
C
C C
U
G G C A G U C A G A G
G
C
G
A
U
G
A
A
G
G
AC
G
U
G
C
UA
A
U
C U
G
C
G
A
U
A
A
G C
G
U
C
G
G
U
A
A
G
G
U
G
A
U
A
U
G
A
A
C
C GU
U
A
UAA
C
C
G
G
C
G
A
U
U
U
C
C
G
A A U G
G
G
G
A A
A
C
C
C A
G
U
G
U
G
U
U
U C
G
A
C
A
C
A
C
U
A
U
C
A
U
U
A
A
C
U
G
A A U
C
C
A
U
AG
G
U
U
A
A
U
G
A
G
G
C
G
A
A
C C G G G G
G A A C
U
G A A
A
C
AUC
UAAGU
A
CCCCGA
G
G
A
A
A
A
G
A
A
AU
C
A
AC
C
G
AGAU
U
C
C
C
C C
A
G
U
A
G
C
G
G
CG
A
G
CG
A
A
C
G
G
G
G
A
G
C
A
G
C
C
C
A
G A G C
C
U G A A
U
C A G U G U G U G U G U U A G U G
G
A
A G
C
G
U
C
U
G
G AA
A
G
G
C
G
C
G
C
G A
U
AC
A
G
G
G
U
G
ACA
G
C
C
C
CG
U
A
CAC
AAA
AAUGCACAUGCUG
UGA
GCUCGAUGA
G
U
A
G
G
G
C
G
G
G
A
C
ACG
U
G
G
U AU
C
C
U
G
U
C
U
G
A
A
U
A
U
G
G
G
G
G
G
A
C C A
U
C
C
U
C
C A A
G
G
C
U
A
A
A
U
A
CU
C
CUGACUG
A
CC
G
A
U
A
GUGAACC
A
G
U
A
CCG
U
G
A G G
G
A
A A G
GCGAAAAGAACCCCGG
C
G A G G G GA GU GAA A A A GAA
CC
U
G
A
A
A
C
C
G
U
G
U
A
C
G
UACAAGCA
G
U
G
G
G
A
G
C
A
C
G
C
UU
A
G
G
C
G
U
G
U
G
A
C
U
G
C
G
U
A C C U UU
U
G
U
AUA
AUGG
GUCAGC
G
A
C
UU
A
U
A
U
U
C
U
G
U
A
G
C
A
A
G G U U
A A
C C G A
A
UAGG
GG
AGCC
G
A
AG
G
G
AA
A
C
C
G
AGUCUUA
A
C
U G G G C G
U
U
A A G
U
U
G
C
A
G
G
G
U
A
U
AG
A
C
C
CG
A
A
AC
C
C
G
G
U
G
A
U
C
U
A
G
C
C
A
U
G
G
G
C
A
G G U U
G A A
G G U U G G G U
A
A
CACUAACU
G
GA
G
GACC
GAA
C
C
G
AC
U
A
A
U
G
ψU
G
A
A
A
A A
U
U
A
G
C
G
G
A
U
G
A
C
U
U
G
U
G
G
C
U
G
G
G
GGU
GA
A
A
G GC
C
A
A
U
C A AA
C
C
G
G
GA
G
A
UA G
C
UG
G
U
U
CUCCCC
G
A
A
A
G
C
U
A
U
U
U
AG
G
U
A
G
CGC
C
U
C
G
U
G
A
A
UU
C
A
U
C
U
C
C
G
G
G
G
G
U
A
G
A
G
C
A
CU
G
U
U
U
C
G
G
C
A
AG
G
G
G
G
U
C
A
UC
C
C
G
A
C
U
U
A C
C
A
A
C
C
C
G
A
U
G
C
A
A
A
C
U
G C
G
A
A
U
A
C
C
G
G
A
G
A A
U
G
U
UA
U
C
A
C
G
G
G
AG
A
C
A
CACGGCGGGψGC
U
A
A C G U C C G U C G U G
A
A
G
A
G
G
G
A
A
A
C A
A
C
C
C
A G A C
C
G
C
C A
G
C
U
A
A
G
G
UCC
C
A AA G
U C
A
U
G
G
U
U
A
A
G
U
G
G
G
A
A
A C
G
A
U
G
U
G
G
G
A
A
G
G
CCC
A
G
A
C A G
C
C
A
G
G
AUGUUGGC
UUA
G
A
A
G C A
G C C A U C A U U
U
A
A
A G
A
A
A
G C
G U
A
A
UA
GCUC
A
C
U
G
G
U
C
G
A
G
U
C
G
G
C
C
U
G
C
G
C
G G A
A
G
A
U
G
U
A
A
C
G
G
G
G
CUAAA
C
C
A
U
G
C
A
C
C
G
A
A
G
C
U
G
C
G
G C
A
G
C
G
A
C
G
C
U U
A
U
G
C
G
U
U
G
U
U
G
G
G
U
A
G G G G A G
C
G
U
U
C
U
G
U
A
A
G
C
C
U
G
C
G
A A G
G
U
G
U
G
C
U
G U
G
A
G
G
C
A
U
G
C
U
G
G
A
G
G
U
A
U
C
A
G
A
AG
U
G C
G
A
A
U
G C U G A C
A
U
A
A
G
U
A
AC
G
A U A A A
G
C
G
G
G
U
G
A
A A
A
G
C
C
C
G
C
U C
G
C
C
G
G
A
A
G
A
C
C
A
A
G
GGUUCCUGUC
CAA
CGU
U
A
A U C G G G G C A G G
G
U
G
A
GU C
G
A
CCCC
UAA
GGC
G
A
G
GCCG
A
A
A G G C
G
U
A
G U C
G A U
G G
G
A
A A
C
A
G
G
U
U
A
A U
A
U
U
C
C
U
G
U
AC
U U G G U G U U A C U G C
G A
A G G G G G
G
A C
G
G
A
G
A
A
G
G
C
U
A
U
G
U
U
G
GCCGGG
CGA
C
G
G
U
U G U
C C C G G U
U
U
A
AGCGU
GUA
GGCUGGUUUUCC
A
GGCA
A
A U C C G G A A A A U C
A A
G G C U
G A G
G C G U G
A
U
G
A C
G A G G C A C U
A
C
GGUGCUGAAGC
A
A
C
A
A
A
U
G
C
C
C
U
G
C
U
U
C
C
A
G
GAAA
A
GCCUCUAAGC
A
UC
A
GGUAACAUCAAA
U
C
G
U
A
C
CC
CAA
A
C C
G A
C
A
CAGGUG
G
U
C A
G G U A G A
G
AAUACC
A
AG
G
C
G C
G
C
U
U
A
A
C
C
U
U
200 400 600 800 1000 1200 1400
-400
-200
0
200
400
The Structure of 16S rRNA
- secondary structure base pair
- tertiary structure base pair
- tertiary structure base triple
a b
c d

Page 6 of 31
ard printing formats, PostScript [http://www.adobe.com/
products/postscript/main.html] and PDF [http://
www.adobe.com/products/acrobat/adobepdf.html] . The
third, named "bpseq," is a simple text format that con-
tains 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, ex-
tent of covariation. These are considered 'tentative' and
are shown on separate 16S and 23S rRNA secondary struc-
ture diagrams [http://www.rna.icmb.utexas.edu/CSI/
2STR/] . These base pairs and base triples have fewer coor-
dinated 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 illus-
trate our system for uniquely and unambiguously num-
bering 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 ver-
sions of the reference structure diagrams replace the nucle-
otides 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 se-
quence 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 with-
Table 1: Reference sequence and nucleotide frequency data available at the CRW Site. Nucleotide frequency data available in tabular
form is indicated with "Y." Entries marked with "*" are also available mapped on the phylogenetic tree. L, Lousy; M, Model; T,
Tentative.
Reference Sequence Single
Nucleotide
Base Pair Base Triple
M T L M T
rRNA
5S Escherichia coli [V00336] Y Y Y
16S Escherichia coli [J01695] Y* Y* Y Y Y* Y
23S Escherichia coli [J01695] V* Y* Y Y Y* Y
tRNA Saccharomyces cerevisiae (Phe) [K01553] Y Y Y
Intron RNA
Group I Tetrahymena thermophila (LSU) [V01416, J01235] Y Y
Group IIA Saccharomyces cerevisiae cytochrome oxidase (mitochondrial)
intron #1 [AJ011856]
Y Y
Group IIB Saccharomyces cerevisiae cytochrome oxidase (mitochondrial)
intron #5 [V00694]
Y Y

Page 7 of 31
in the boundaries of another helix, forming large cooper-
ative sets of nested helices. The remaining base pairs form
tertiary interactions that either span two sets of nested hel-
ices, forming a pseudoknot, or are involved in base triple
interactions.
In the "Matrix of All Possible Helices" plot [http://
www.rna.icmb.utexas.edu/CSI/2STR/] , the same RNA se-
quence is extended along the X- and Y-axes, with all po-
tential 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 ac-
tual number present in the biologically-active structure
(see CRW Methods [http://www.rna.icmb.utexas.edu/
METHODS/] ). For example, the S. cerevisiae phenyla-
lanine 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 approxi-
mately 2.5 × 1019; there are approximately 10393 and
10740 possible structure models for 16S and 23S rRNA, re-
spectively (see CRW Methods [http://www.rna.ic-
mb.utexas.edu/METHODS/] ). Covariation analysis
accurately predicted the structures of the 16S and 23S rR-
NAs (see above) from this very large number of structure
models.
Table 2: Alignments available from the CRW Site. These alignments were used to generate conservation diagrams (rRNA only) and
correspond to the alignments used in the nucleotide frequency tables.
Molecule Alignment # of Sequences
rRNA (5S / 16S / 23S) T (Three Domains/Two Organelles) 686/6389/922
3 (Three Phylogenetic Domains) -- / 5591 / 585
A (Archaea) 53/171/39
B (Bacteria) 323/4213/431
C (Eukaryota chloroplast) -- / 127 / 52
E (Eukaryota nuclear) 299/1937/115
M (Eukaryota mitochondria) -- / 899 / 295
Group I Intron A (IA1, IA2, and IA3 subgroups) 82
B (IB1, IB2, IB3, and IB4 subgroups) 72
C (IC1 and IC2 subgroups) 305
Z (IC3 subgroup) 125
D (ID subgroup) 19
E (IE subgroup) 46
U (all other group I introns) 41
Group II Intron A (IIA subgroup) / B (IIB subgroup) 171/571
tRNA A (Alanine tRNAs) / C (Cysteine tRNAs) 64/19
D (Aspartic Acid tRNAs) / E (Glutamic Acid tRNAs) 35/49
F (Phenylalanine tRNAs) / G (Glycine tRNAs) 54/69
H (Histidine tRNAs) / I (Isoleucine tRNAs) 38/56
K (Lysine tRNAs) / M (Methionine tRNAs) 53/36
N (Asparagine tRNAs) / P (Proline tRNAs) 35/55
Q (Glutamine tRNAs) / R (Arginine tRNAs) 35/62
T (Threonine tRNAs) / V (Valine tRNAs) 49/65
W (Tryptophan tRNAs) / X (Methionine Initiator tRNAs) 30/65
Y (Tyrosine tRNAs) / Z (All Type 1 tRNAs) 47 / 895

Page 8 of 31
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 iden-
tified 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 Brima-
combe [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 new-
er interactions proposed here.
This analysis produced two very large tables with 579 pro-
posed 16S rRNA base pairs evaluated against six versions
of the structure model and 1001 23S rRNA base pairs eval-
uated against five versions of the structure model. Some
highlights from these detailed tables are captured in sum-
mary tables (Tables 3a and 3b, and [http://www.rna.ic-
mb.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 cur-
rent structure model is considered to be the correct struc-
ture; thus, values for comparisons are referenced to the
numbers of sequences and base pairs in the current struc-
ture model (478 base pairs and approximately 7000 se-
quences 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 cur-
rent 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 pro-
posed base pairs that are not in the most current structure
models (H-1B.2).
An analysis of these tables reveals several major conclu-
sions from the evolution of the 16S and 23S rRNA covari-
ation-based structure models. First, approximately 60% of
the 16S and nearly 80% of the 23S rRNA base pairs pre-
dicted in the initial structure models appear in the current
structure models. The accuracy of these early models, pro-
duced from the analysis of only two well-chosen sequenc-
es, is remarkable. Second, the accuracy, number of
Table 3a: Summary of the Evolution of the Noller-Woese-Gutell 16S rRNA Comparative Structure Model. Categories marked with "*"
are calculated compared to the 1999 version of the 16S rRNA model.
Date of Model 1980 1983 1984–86 1989–90 1993–96 Current (1999)
1. Approximate # Complete Sequences 2 15 35 420 1000 7000
2.% of 1999 Sequences 0.03 0.2 0.5 6.0 14.3 100
3. # BP Proposed Correctly * 284 388 429 450 465 478
4. # BP Proposed Incorrectly * 69 49 38 28 6 0
5. Total BP in Model (#3 + #4) 353 437 477 478 471 478
6. % of BP in This Model that Appear in the Current Model (#3 / 478) * 59.4 81.2 89.7 94.1 97.3 100
7. Accuracy of Proposed BP (#3 / #5) 80.5 88.8 89.9 94.1 98.7 100
8. # BP in Current Model Missing from This Model (478 - #3) * 194 90 49 28 13 0
9. # Tertiary BP Proposed Correctly * 4 8 15 25 35 40
10. % Tertiary BP Proposed Correctly * 10.0 20.0 37.5 62.5 87.5 100
11. # Base Triples Proposed Correctly * 0 0 0 0 0 6
12. % Base Triples Proposed Correctly * 0 0 0 0 0 100

Page 9 of 31
secondary and tertiary structure interactions, and com-
plexity of the structure models increase as the number and
diversity of sequences increase and the covariation algo-
rithms 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 posi-
tional 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 Defini-
tions 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 he-
lix (two or more consecutive, antiparallel and nested base
pairs) or a tertiary interaction, which is a more heteroge-
neous 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 ar-
rangement, and base triples (a single nucleotide interact-
ing with a base pair). Each of these base pair categories has
a unique color code in the illustrations on the "RNA Struc-
ture 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 struc-
ture models. Within this category, they can be within a
hairpin loop (nucleotides capping the end of a helix), in-
ternal loop (nucleotides within two helices), or in a multi-
stem loop (nucleotides within three or more helices).
2. Nucleotide frequency and conservation information
Underpinning the comparative sequence analysis of RNA
molecules are the realizations that every RNA has evolved
to its present state and form, and that the same secondary
and tertiary structure for an RNA can be derived from
many different sequences that maintain the integrity and
functionality of that structure. These evolutionary and
structural dynamics have made it possible to predict RNA
structure models with comparative analysis (as presented
in the previous section). The tempo and mode of the evo-
lution for every position in the RNA structure is defined by
a complex and not-well-understood equation, with varia-
bles for global mutation rates and rates for specific
branches on the phylogenetic tree, the allowed variance
for each nucleotide and the structure with which it is asso-
ciated, the coordination and dependence between nucle-
otides, and other constraints not yet defined. In an effort
to begin to understand these dimensionalities associated
with an RNA sequence and to catalogue the observed con-
straints in each of the RNA molecules maintained within
our CRW Site, we have prepared online tables and figures
that reveal the amount and type of conservation and vari-
ation for many of the RNAs available here.
The comparative information for a sequence is initially as-
sembled in a sequence alignment (more information
about alignments below at: "3. Sequence and Structure
Data"). The extent and type of sequence and structure con-
servation and variation are presented in two general for-
mats: (1) nucleotide frequency tables that contain the
types of nucleotides and their frequencies for each posi-
Table 3b: Summary of the Evolution of the Noller-Woese-Gutell 23S rRNA Comparative Structure Model. Categories marked with "*"
are calculated compared to the 1999 version of the 23S rRNA model.
Date of Model 1981 1984 1988–90 1992–96 Current
(1997–2000)
1. Approximate # Complete Sequences 2 15 55 220 1050
2.% of 1999 Sequences 0.2 1.4 5.2 21.0 100
3. # BP Proposed Correctly * 676 692 794 836 870
4. # BP Proposed Incorrectly * 102 93 69 26 0
5. Total BP in Model (#3 + #4) 778 785 863 862 870
6. % of 1999 Model Proposed Correctly (#3 / 870) * 77.7 79.5 91.3 96.1 100
7. Accuracy of Proposed BP (#3 / #5) 86.9 88.2 92.0 97.0 100
8. # BP in Current Model Missing from This Model (870 - #3) * 194 178 76 34 0
9. # Tertiary BP Proposed Correctly * 4 3 29 49 65
10. % Tertiary BP Proposed Correctly * 6.2 4.6 44.6 75.4 100
11. # Base Triples Proposed Correctly * 0 0 0 2 7
12. % Base Triples Proposed Correctly * 0 0 0 28.6 100

Page 10 of 31
tion in the RNA molecule; and (2) secondary structure di-
agrams revealing the most conserved nucleotide at each
position that is present in the vast majority of the sequenc-
es. The position numbers for the nucleotide frequency ta-
bles and conservation diagrams are based upon a
reference sequence (see Table 1). While deletions relative
to the reference sequence are shown in the tables with "-,"
insertions relative to the reference sequence are not
shown. Conservation diagrams summarize the insertions
and deletions relative to the reference sequence.
2A. Nucleotide frequency tabular display
The nucleotide frequency tables appear in two general
presentation modes. In the traditional table, the nucle-
otide types are displayed in the columns, while their fre-
quencies are shown for each alignment in the rows. The
nucleotide frequencies were determined for single posi-
tions, base pairs, and base triples for a subset of the RNAs
in the CRW Site collection (detailed in Table 1). Single nu-
cleotide frequencies are available for all individual posi-
tions, 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 com-
parative analysis that are not included in our current struc-
ture 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 nu-
cleotide; 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 align-
ments, where the most similar sequences are organized
into the same alignment. For the three rRNAs, the align-
ments 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 Eucar-
ya organelles (no alignments yet for the 5S rRNA; (4)
Chloroplasts and (5) Mitochondria), and two larger align-
ments that include all of the (6) nuclear-encoded rRNA se-
quences for the Archaea, Bacteria, and Eucarya, and (7)
these three phylogenetic groups and the two Eucarya or-
ganelles (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 func-
tional 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 includ-
ed 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 clas-
sifications 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 un-
known. The group II introns are divided into the two ma-
jor 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 frequen-
cy 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 confi-
dence rating for the base pairs predicted with the covaria-
tion 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 de-
crease in the number of single uncompensated changes,
and the number of times these coordinated variations oc-
cur during the evolution of that RNA (for the rRNAs, the
number of times this covariation occurs in the phyloge-
netic 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. Final-
ly, blue base pairs do not satisfy these constraints; never-
theless, we are confident of their authenticity due to a
significant number of covariations within the sequences

Page 11 of 31
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 in-
dependently 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 rank-
ing 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 confi-
dence) column. For this base pair, only the T (Three Phy-
logenetic 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 align-
ment, while the C1+C3 score is 1.00, greater than the min-
imum 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.utex-
as.edu/METHODS/]). Note that the 928:1389 and
929:1388 base pairs are also both red. Here, six of the sev-
en alignments have significant extents of covariation for
both base pairs and are thus red. Each of the red align-
ments 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 phy-
logenetic tree [http://www.ncbi.nlm.nih.gov/Taxonomy/
taxonomyhome.html/] [60,61] (see Materials and Meth-
ods 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 availa-
ble 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 initial-
ly reveal, in the larger section of the main frame, the root
of the phylogenetic tree, with the frequencies for the se-
lected single base, base pair, or base triple. The presenta-
tion for single bases (H-2B.1) reveals the nucleotides and
their frequencies for all sequences at the root level, fol-
lowed by the nucleotides and their frequencies for the Ar-
chaea, 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 "per-
centage 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 but-
ton 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 Eur-
yarchaeota, and four of these are within the Halobacteri-
ales (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 rep-
resentative 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 re-
gion). 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

Page 12 of 31
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 in-
sertion 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 dia-
gram (H-2C.1). All insertions greater than five nucleotides
are tabulated, in addition to insertions of one to four nu-
cleotides 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/taxonomy-
home.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 Bac-
teria; (5) the Eucarya (nuclear encoded); (6) the chloro-
plasts; 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 ma-
jor 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 ta-
ble 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 se-
quences 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 sci-
entific name); 2) GenBank accession number; 3) cell loca-
tion; 4) RNA Type; 5) RNA Class; and 6) NCBI phylogeny.
Users who want more information about a given se-
quence 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 struc-
ture
Analysis of the patterns of sequence conservation and var-
iation 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 align-
ment. These alignments are an attempt to approximate
the best juxtapositioning of sequences that represent sim-
ilar 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 iden-
tical subsequences with one another. However, when
there is a significant amount of variation between the se-
quences, 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.). Giv-
en the accurate prediction of the 16S and 23S rRNA sec-
ondary structures from the analysis of the alignments we
assembled, we are now even more confident in the accu-
racy 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 align-
ments, 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 phyloge-
netic domains and the two eucaryotic organelles, the chlo-
roplast and mitochondria. Other regions of the rRNA are
conserved within the three phylogenetic domains al-
though variable in the mitochondria. As we proceed into
the phylogenetic tree, we observe positions that are con-
served 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 pat-
terns of conservation and variation transcend all levels of
the phylogenetic tree and result in features in the rRNA se-
quences 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

Page 13 of 31
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 dependen-
cies, 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 collec-
tion also contains many structural variations, in addition
to their conserved sequence and structure core. The major-
ity 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 read-
ily 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 po-
sitional 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 phylogenet-
ic 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 pre-
dict 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 Eucaryo-
tic 23S-like rRNA sequences was determined, we reana-
lyzed these two regions and were able to align those
regions to other related organisms (e.g., S. cerevisiae with
Schizosaccharomyces pombe, Cryptococcus neoformans, Pneu-
mocystis carinii, Candida albicans, and Mucor racemosus)
and predict a secondary structure that is common for all
of these rRNAs (see Figures 3 and 6 in [52]). While the sec-
ondary structures for the fungal 23S-like rRNAs are deter-
mined in these regions, the animal rRNAs were only
partially solved. We still need to determine a common sec-
ondary 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 se-
quences that represent the major phylogenetic groups,
and for those sequences that reveal the major forms of se-
quence and structure conservation and variation. New sec-
ondary structure diagrams are templated from an existing
secondary structure diagram and the alignment of these
two sequences, the sequence for the new structure dia-
gram and the sequence for the structure that has been tem-
plated. 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 se-
quence 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 dia-
grams occurs in parallel with the development of the se-
quence alignments. In some cases, the generation of a
structure diagram helps us identify problems with the se-
quence or its alignment. For example, anomalies in struc-
tural elements (in the new structure diagram) that had
strong comparative support in the other sequences could
Figure 3
RDBMS (Standard) search form.

Page 14 of 31
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 re-
gion that was weakly predicted with comparative analysis.
However, a re-inspection of a few related structure dia-
grams revealed another potential helix in this region that
was then substantiated from an analysis of the corre-
sponding region of the alignment. Thus, the process of
generating additional secondary structure diagrams im-
proves the sequence alignments and the predicted struc-
tures, 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 struc-
ture 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 gen-
erally has higher priority over other rRNA sequences.
C) Enter pertinent information for each sequence and
structure into our relational database management sys-
tem. This computer system organizes all of our RNA se-
quence and structure entries, associates them with the
organisms' complete NCBI phylogeny [http://www.nc-
bi.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 de-
termination of nucleic acid sequence information, the
number of ribosomal RNA and group I and II intron se-
quences 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 se-
quences 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 dia-
grams 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 availa-
ble sequences for the Archaea, Bacteria, and Eucarya nu-
clear, 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 se-
quences and structures for more expanded lists for each of
those five phylogenetic/cell location groups. For example,
the Archaea are expanded to the Crenarchaeota, Euryar-
chaeota, 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 di-
agrams 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 alphabet-
ically. 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 oth-
er) 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 ap-
proximately twice as many group I introns than group II
introns. Of the 406 secondary structure diagrams, the ma-
jority 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). Dia-
grams are listed alphabetically by organism name within
each of the three time categories. The display also indi-
cates 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 download-
ed from the Secondary Structure Retrieval Page (Highlight
3C.1). This system allows the user to select from organism

Page 15 of 31
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 for-
mats. Once these selections are made, a list of the struc-
ture 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 pre-
sented on one line as a single unit to ensure that both
halves are downloaded. The system packages the second-
ary 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 align-
ments might also contain refinements in the alignments
of the sequences. For each alignment, there is a corre-
sponding list of sequences, their phylogenetic placement,
and other information about the sequences (see conserva-
tion 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 avail-
able in June 2002.
Table 4: RDBMS Fields and Short Descriptions.
# Search Query Output Field Description
1 ---- Row# Index for ease of usage.
2 Organism Organism Organism: Complete organism name (in Genus species format; organisms are listed using the
NCBI scientific name).
3 Cell Location L Cell Location: Chloroplast (C), Cyanelle (Y), Mitochondrion (M), Nucleus (N), or Virus (V).
4 RNA Type RT RNA Type: rRNA (R) or Intron (I). (mRNA, tRNA, SnRNA, and Other are presently unsup-
ported.)
5 RNA Class RC RNA Class: Detailed classification within RNA Types.
6 Exon EX Exon sequence containing the intron. The expanded names for the exon abbreviations are availa-
ble online.
7 ---- IN Intron Number: For exon sequences containing multiple introns, the introns are numbered
sequentially.
8 Intron Position IP Intron Position: Nucleotide (E. coli reference numbering) immediately prior to the intron inser-
tion point.
9 ORF 0 Open Reading Frame presence within intron sequences. Y = an ORF of at least 500 nucle-
otides is present; N = no ORF of at least 500 nucleotides is present; U = ORF presence/absence
was not determined; see also online discussion about ORFs. The ORF identity is sometimes given
in the Comment field.
10 Sequence Length Size Number of nucleotides in the RNA sequence.
11 ---- Cmp Percent Completeness: estimated completeness of the sequence. Only sequences that are at
least 90% complete are included here.
12 Accession Number AccNum GenBank Accession Number. Links directly to the GenBank entry at the NCBI web site.
13 Secondary Struc-
tures
StrDiags Structure Diagrams: Links to secondary structure diagrams available from the CRW Site.
Users may select sequences with or without structures or all sequences.
14 Common Name Common Name From the NCBI Phylogeny, where available.
15 Group ID Gr.Id (Partially implemented feature.)
16 Group Class Gr.Class (Feature not presently implemented.)
17 Comment Comment Additional information about a sequence.
18 Phylogeny Phylogeny NCBI Phylogeny for the Organism. The first level is shown; the remainder is available by following
the "m" ("more") link.
19 ---- Row# Index for ease of usage.
#: order of appearance of fields in the RDBMS output. Search Query: names of fields on the Search screen; ----, not available as a search criterion.
Output Field: names of fields in the RDBMS output. Description: more information about the field and its contents. The RDBMS Search page
contains two additional options: Results / Page, which allows users to display 20, 50, 100, 200, or 400 results per page, and Color Display, which
toggles alternating colored highlighting of adjacent organisms. Expanded descriptions of each field and the corresponding contents are available
online at the CRW RDBMS Help Page.

Page 16 of 31
3E. rRNA Introns
The introns that occur in 16S and 23S rRNAs are organ-
ized 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 oc-
curs between the position number listed and the follow-
ing position (e.g., the introns between position 516 and
517 are listed as 516). rRNA Introns Table 1 has four com-
ponents.
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, sort-
ed by the numeric order of the intron positions, is con-
tained 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 (min-
imum # – 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: Phyl-
ogenetic Distribution); and 9) the organism name and ac-
cession 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 Decem-
ber 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 phylo-
genetic groups, etc.
Additional information is available in a new window for
each of the values in rRNA Introns 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) acces-
sion number; 9) link to structure diagram (if it is availa-
ble); and 10) comment.
The number of intron types per intron position are tabu-
lated 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 in-
trons per rRNA position; 2) number of positions with that
number of introns; 3) the rRNA position numbers; 4) to-
tal 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 ob-
served number of introns per rRNA site.
The highlights from rRNA Introns Table 1 are: 1) As of De-
cember 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 rR-
NA. 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). In-
stead, nearly 50% (71/152) of the intron positions con-
tain 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 in-
trons (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 in-
trons with the Poisson distribution for the observed
number of introns. The Poisson distribution, P(x) = e-µ
µxx!-1, where µ is the mean frequency of introns for posi-
tions in a particular exon and x is the target number of in-
trons 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 ex-
pect 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 posi-
tions 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 availa-
ble insertion positions is extremely unlikely to occur by
chance. 5) While a single intron type occurs at the major-
ity of the intron positions, several positions have more
than one intron type. A few of the positions that deserve

Page 17 of 31
special attention have IC1 and IE introns at the same po-
sition (16S rRNA positions 516 and 1199, and 23S rRNA
position 2563). The 16S rRNA position 788 has several ex-
amples 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 in-
tron 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 in-
trons 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 in-
trons), 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%) oc-
cur 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 "Un-
classified" 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 with-
in the "Unclassified" category. The first is a series of 43 in-
trons 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 nucle-
otides. The second group contains 121 spliceosomal in-
trons 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 splice-
somal introns occur only in nuclear rRNA genes and tend
to occur at unique sites; the lone exception is the spliceo-
somal intron from Dibaeis baeomyces nuclear 23S rRNA
position 787, a position where a group IIB intron occurs
in mitochondrial Marchantia polymorpha rRNA. The Un-
classified 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 provid-
ing links to twenty additional tables (H-3E.7), each of
which provides expanded information about a specific in-
tron type. The organism name, exon, intron position, cell
location, and complete phylogeny are accessible for each
intron from these tables. These online tables are dynami-
cally 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 tab-
ulated 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 distri-
butions are: 1) The majority (96%) of the rRNA introns
occur in Eucarya, followed by the Archaea, and the Bacte-
ria. 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 fre-
quency of introns in Archaea rRNAs is higher, with 43 ex-
amples documented as of December 2001. Within the
Archaea, there is a higher ratio of rRNA introns in the Des-
ulfurococcales and Thermoproteales subbranches in the
Crenarchaeota branch. 4) For the three primary phyloge-
netic 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 nucle-
ar-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 se-

Page 18 of 31
quences are an underestimate, since many rRNA introns
are published with only short flanking exon sequences
and do not meet the 90% completeness criterion for in-
clusion 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 lo-
cation 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 in-
trons. 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 classifi-
cations include a link to the complete details (organism
name, phylogeny, cell location, exon, intron position, in-
tron 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 docu-
mented 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 in-
trons among their exon sequences, the Poisson distribu-
tion (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 in-
trons (rRNA Introns Table 4). Based upon this data, no
rRNA sequences are expected to contain four or more in-
trons; 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 con-
taining large numbers of introns. In 16S rRNA, only nucle-
ar 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 mito-
chondrial). 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 com-
prising the remaining eight sequences.
rRNA Introns Table 4 provides access to seventeen addi-
tional 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 pub-
lished 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 sta-
tistical 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. "Phy-
logeny" (and "Cell Location;" H-3F.1). Group I and II in-
tron data are highlighted with yellow and blue
backgrounds, respectively. The phylogenetic divisions are
also split into the three possible cellular locations (nucle-
ar, chloroplast, and mitochondria). A few of the high-
lights 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 chloro-
plast and mitochondrial genes.
Intron Distribution Table 2 shows "Intron Type" vs. "Ex-
on" (and "Cell Location;" H-3F.2). Again, group I and II
intron data are highlighted with yellow and blue back-
grounds, respectively. In this table, the exon types are split
into the three possible cellular locations (nuclear, chloro-
plast, and mitochondria). As of December 2001, the most
obvious trend is that the exons with the most Group I in-

Page 19 of 31
trons are 16S rRNA (900), leucine tRNA (337), 23S rRNA
(284), ribosomal protein S16 (214), and ribosomal pro-
tein 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 cel-
lular 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 De-
cember 2001, links connecting to the specific RDBMS re-
sults are not available.
4. Data access systems
For our first generation of online comparative RNA struc-
ture databases (16S rRNA [46,47], 23S rRNA [48–52], and
group I Intron [33]), we organized the rRNA and group I
intron secondary structures into a simple static set of man-
ually-generated HTML pages. The structure diagrams were
organized first by RNA type (for the rRNAs; e.g., all 16S
rRNA diagrams were grouped together) or structural sub-
type (for group I introns; e.g., IC1) and then by the phyl-
ogenetic order of the organisms. This type of presentation
is acceptable, although not ideal, for a small number of
entries. However, it is grossly inadequate and inefficient
for larger numbers of entries and more fields of informa-
tion. Thus, with the anticipation that our database of com-
parative RNA information would grow significantly, the
need to associate more fields of information with each en-
try, to automatically and dynamically generate the HTML
output for all queries of the database, and the ability to
search our database for entries with specific attributes in
many fields and to sort those fields in the output with dif-
ferent priorities, we have developed a relational database
management system (RDBMS) that is built on the MySQL
database program (see Materials and Methods).
Our goal was to create a system that would allow for the
following examples of dynamic searches of our CRW RD-
BMS. Find and output:
A. Homo sapiens 5S, 16S, and 23S rRNA entries.
B. Enteric bacterial rRNA sequences and/or secondary
structure diagrams.
C. 1) Tunicate and 2) Coelacanth rRNA sequences.
D. All 23S rRNA sequences. Sort output by four methods:
1) organism name, alphabetically; 2) phylogenetic classi-
fication; 3) sequence length; and 4) first by cellular loca-
tion, then by phylogenetic classification.
E. Group I introns that occur: 1) in Saccharomyces cerevi-
siae, 2) in mitochondria, 3) in the exons 16S and 23S rR-
NA, 4) at position 516 in 16S rRNA, 5) in the IE subgroup,
6) in the IE subgroup at 16S rRNA position 516.
Each sequence and structure entry has the following fields
or attributes: organism name, NCBI phylogeny, common
name, cell location, RNA type, RNA class, sequence
length, accession number, intron number, intron posi-
tion, exon, open reading frame, link to secondary struc-
ture diagram (if it exists), and comment. An abbreviated
explanation for each of these attributes is given in Table 4;
a full explanation is available online at the RDBMS page.
The RDBMS and the data that it contains are accessed by
several different graphical interfaces. One interface, the
"Index of Available RNA Sequences and Structures," was
described in Section 3, "Sequence and Structure Data."
The SQL queries on this page were predetermined and re-
stricted. The "Index" contains the number of sequences
and structures for different molecules and phylogenetic
groups. Clicking a link searches the current database for
all entries that satisfy that specific query (e.g., bacterial
16S rRNA structures) and dynamically generates the out-
put. The SQL queries for Sections 3E (rRNA Introns) and
3F (Group I/II Intron Distribution) are also preset. In con-
trast with the predetermined and restricted searches avail-
able on these pages, we have also developed two different
graphical interactive interfaces for Section 4, "Data Access
Systems," that allow the user to define and implement
their own search of the same information in our relational
database management system. The first one, called
"Standard," is the least restrictive and allows the user to
search for any values present in one or a combination of
the attributes and to sort the output on any combination
of attributes (see Section 4A below). The second one is
semi-restrictive and allows the user to navigate through
the phylogenetic tree to search for those entries that are
within specific phylogenetic groups (see Section 4B be-
low).
4A. RDBMS (Standard)
The "Standard" interface is the most fundamental of our
interfaces to the CRW RDBMS information. While the re-
stricted, specialized interface to the RDBMS information
in Section 3A requires minimal instruction to use, the
standard interface, with its ability to cull out all arrange-
ments of information from the different fields with so-
phisticated 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

Page 20 of 31
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 availa-
ble from the links to the attribute names. This informa-
tion 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 dis-
played on the screen and then clicks the "Go" button.
While the user can select the individual fields (e.g., "Or-
ganism" or "Phylogeny"), for most applications the "QrR-
NA" (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 accept-
able 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 deter-
mined with the "V" (values) button on the right side of
the main frame; the results are displayed in the right frame
(see Figure 3 and 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 read-
ing 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, Se-
quence 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" but-
ton 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 "Or-
ganism," "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 set-
tings 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 phyloge-
netic 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 op-
eration.
For mode one, you can systematically navigate through
the phylogenetic tree to the selected goal point. For exam-
ple, to get to the last phylogenetic group that contains
Homo sapiens and gorillas, the user would click on the "Eu-
karyota" phylogeny button, then click on the "Fungi/
Metazoa group" link in the right frame, followed by the
"Metazoa," "Eumetazoa," "Bilateria," "Coelomata," "Deu-
terostomia," "Chordata," "Craniata," "Vertebrata," "Gna-
thostomata," "Teleostomi," "Euteleostomi,"
"Sarcopterygii," "Tetrapoda," "Amniota," "Mammalia,"
"Theria," "Eutheria," "Primates," "Catarrhini," and "Ho-
minidae" links. The phylogenetic group Hominidae con-
tains 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

Page 21 of 31
help you jump to the appropriate node in the phylogenet-
ic 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 3 and H-4A.1).
The "Common Name" attribute can also help identify or-
ganism 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 pre-
sumed 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 pole-
cat (Mustela putorius), and duckbill platypus (Ornithorhyn-
chus 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 intend-
ed to identify larger groups of organisms. The three but-
tons 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 ar-
ranged 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 organ-
isms 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, Eu-
karyota, Fungi/Metazoa group, Metazoa, Eumetazoa, Bila-
teria, 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. Select-
ing "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 Mam-
malian or Bryophyta phylogenetic groups. Thus, the com-
mon 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 phylog-
eny 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 answer-
ing questions easier. Take, for example, a search for all Eu-
carya 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 <or-
ganism name, RNA class, phylogeny, and cell location>
produce significantly different orders and overall arrange-
ments 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 phylo-
genetic 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 3 and 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 al-
phabetical/numerical order for any attribute can be re-
versed (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 de-
fault 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 se-
quence 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 en-
tries with a secondary structure, or entries without a sec-
ondary 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 select-
ing the "Next" button on the left bottom frame in the out-
put 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

Page 22 of 31
white) alternate for changes in the organism names in the
output entries.
• Group ID and Group Class: these two attributes are cur-
rently 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 in-
dicates the number of entries shown in the window and
the entry numbers currently shown, and, if necessary, con-
tains 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 on-
line help from the "Secondary Structure" and "StrDiags"
links on the RDBMS query and results pages) can be re-
trieved 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 NC-
BI. 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 se-
quence.
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 con-
tains the known common names associated with each lev-
el 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 de-
veloped to facilitate the identification and retrieval of se-
quence 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 inter-
face displays the entire phylogenetic tree, including those
branches that do not have corresponding entries. This in-
terface is based on the Taxonomy Browser developed by
NCBI [http://www.ncbi.nlm.nih.gov/Taxonomy/taxono-
myhome.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 4 and 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 infor-
mation 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 lev-
els 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
Figure 4
RDBMS (PhyloBrowser) basic phylogenetic search screen,
showing two additional levels of phylogeny.

Page 23 of 31
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 4 and 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 ap-
propriate 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 phyloge-
netic 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 Re-
sults 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 in-
trons) 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 individ-
ual 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 RD-
BMS 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 se-
quences 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 avail-
able 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 "Ac-
knowledgement"). Additional information for each of
these attributes is then displayed in a new window by
clicking on either the attribute link or the additional infor-
mation link in the top-right frame (Figure 4 and 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 struc-
tural elements. Given this limitation, we have utilized
comparative analysis to determine that RNA structure that
is common to a set of functionally and structurally equiv-
alent 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 pre-
dicting these comparative structure models, we have de-
termined a large number of 5S, 16S, and 23S rRNA and
group I intron comparative structure models from se-
quences that are representative of all types of structural
variations and conservation. Thus, with the correct rRNA
structure models and a large sampling of structurally di-
verse structure models, we now want to decipher more re-
lationships 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 struc-
ture 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 covari-
ation-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 ade-
nosines (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 5 and H-4C.

Page 24 of 31
This RNA sequence/structure query system has three pri-
mary fields of input to be selected by the user: the RNA
type, phylogenetic group/cell location, and the nucle-
otide/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 in-
trons) and five phylogenetic groups/cell locations (Bacte-
ria, Archaea, Eucarya nuclear-encoded, mitochondrial,
and chloroplast). Any combination and number of RNA
types and phylogenetic/cell location groups can be select-
ed, 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 5 and
H-4C. Five nucleotide categories are searchable: single nu-
cleotides, (two) adjacent nucleotides, base pairs, three nu-
cleotides, 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 nu-
cleotide 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 select-
ed structural elements, the most frequent nucleotide pat-
tern, followed by other patterns in descending order to the
least frequent nucleotide pattern. For the "A Story" exam-
ple mentioned earlier, adenosine is the most frequent nu-
cleotide at unpaired positions (42.64%), followed by G
(23.6%), U (21.27%), and C (12.49%) (Figure 5 and 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 5 and 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 percentag-
es 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 dinu-
cleotides are AA (19.2%; H-4C.3), followed by GA
(13.35%), UA (9.821%), AU (6.703%), etc. The most fre-
quent 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 se-
quences 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 nu-
cleotides reveal several strong biases in the distribution of
nucleotides in different structural environments.
The top section of the output page (Figure 5 and H-4C.1)
displays the types of data (RNA molecules and phyloge-
netic/cell location groups) that were selected and ana-
lyzed. This section also reveals the number of structure
models that were analyzed; 175 16S and 71 23S rRNA
structure models were analyzed in Figure 5 and H-4C.
A few of the other biases in the distribution of nucleotide
patterns that were determined with this sequence/struc-
ture 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 else-
where. The "Structure, Motifs, and Folding" section pres-
ently contains three RNA motif projects ("U-Tum" [62],
"A Story" [63], and "AA.AG@helix.ends" [64]) and two
RNA folding projects ("16S rRNA Folding" [65] and "23S
rRNA Folding" [66]). In the "Phylogenetic Structure Anal-
ysis" section, additional information for three publica-
tions is available: "Mollusk Mitochondria" [67], "Polytoma
Leucoplasts" [68], and "Algal Introns" [69].
Figure 5
Analysis of the Bacterial 16S and 23S rRNA structure models
using the "RNA Structure Query System." The entire system
(selection frame and results) is shown with the results for
the distribution of single nucleotides, sorted in order of
decreasing prevalence in unpaired regions.

Page 25 of 31
Table 5: Attributes for the "RNA Structure Query System." The 5' and 3' ends of helices and loops are based on the global orientation
determined from the 5' and 3' ends of the entire RNA molecule.
RNA Types 5S rRNA, 16S rRNA, 23S rRNA, Group I intron
Phylogenetic Groups / Cell Locations Bacteria (nucleus), Archaea (nucleus), Eucarya (nucleus, mitochondria, and chloroplast)
number/type of nucleotides structural element short name brief explanation (if necessary)
single nuc total
paired (helix) paired positions
unpaired (loop) unpaired positions
5' helix end 5' end of helix
3' helix end 3' end of helix
5' loop end 5' end of loop
3' loop end 3' end of loop
helix center in helix but not at the 5' or 3' ends
loop center in loop but not at the 5' or 3' ends
unpaired/paired ratio of 'unpaired' / 'paired'
adjacent nucs total
in helix paired positions
in loop unpaired positions
3'helix 5'loop junction: 3' end ofhelix/5' end of loop
3'loop 5'helix junction: 3' end ofloop/5' end of helix
in loop/in helix ratio 'in loop' / 'in helix'
base pairs total
5'helix end at the 5' end of a helix
3'helix end at the 3' end of a helix
helix center in helix, but not at the 5' or 3' ends
three nucs total
000, 111,001,011,010, 100, 101,110 0 = unpaired, 1 = paired; patterns of three con-
secutive nucleotides
5'-(A:C)B base pair with an unpaired nucleotide 3' to one
paired position
5'-A(B:C) base pair with an unpaired nucleotide 3' to one
paired position
four nucs total
0000,1111,0001, 1110,0010,
1101,0011,1100,0100, 1011, 0101,1010,0110,
1001, 1000, 0111
0 = unpaired, 1 = paired; patterns of four consec-
utive nucleotides
double pair@5end two consecutive base pairs at the 5' end of heli-
ces
double pair@mid two consecutive base pairs not at the 5' or
3'ends of helices
double pair@3end two consecutive base pairs at the 3' end of heli-
ces
5-(A:D)BC base pair with two consecutive unpaired nucle-
otides 3' to one paired position
lonepair base pair with unpaired nucleotides 5' and 3' to
one unpaired position
5-AB(C:D) base pair with two consecutive unpaired nucle-
otides 5' to one paired position

Page 26 of 31
Conclusions
Nearly 10 years ago, our initial goals for our RNA web
page was to disseminate some of the comparative infor-
mation 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 con-
ditions of the search. In parallel, with the significant ad-
vancements 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 posi-
tions in the reference structure;
3) secondary structure conservation diagrams that reveal
the extent of conservation in the RNA sequence and struc-
ture;
4) representative secondary structure diagrams for organ-
isms 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 rR-
NA, 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 manage-
ment 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 ele-
ments, and B) increases in the number of aligned sequenc-
es; 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 anal-
ysis; 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 informa-
tion and new modes of presenting the data; and 8) ana-
lyze more types of RNA molecules from a comparative
perspective, and present this data in the same formats uti-
lized 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 align-
ments 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/Ent-
rez/] at least once per week with appropriate search crite-
ria (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 availa-
ble 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 en-
tered into the appropriate sequence alignment. A few se-
quences 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 RD-
BMS.
Deviations in GenBank entries
The majority of GenBank entries contain accurate annota-
tions of the RNAs. However, some GenBank entries devi-
ate 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 char-
acteristic of that rRNA (a few examples). Sometimes, in-
tron sequences are not annotated and were discovered
during the alignment of the corresponding rRNA exons
(e.g., the unannotated intron in the uncultured archaeon

Page 27 of 31
SAGMA-B 16S rRNA (AB050206) and many Fungi, in-
cluding AF401965 [88]). Other GenBank entries contain
incorrect annotations for the RNAs; the boundaries may
be misidentified by a small or large number of nucle-
otides.
RNA sequence alignment and classification of intron se-
quences
Alignment and determination of intron-exon boundaries
The sequence alignments used for this analysis are main-
tained 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
Table 6: Significant values from the "RNA Structure Query System." The 5' and 3' ends of helices and loops are based on the global
orientation determined from the 5' and 3' ends of the entire RNA molecule. Values are for the Bacterial 16S and 23S rRNA comparative
structure models.
Number/Type of Nucleotides Structural Element
Short Name High Low
single nuc total
paired (helix) G (36.57%) A (14.46%)
5' helix end G (46.23%) U(13.52%)
3' helix end C (38.07%) A (10.57%)
5' loop end G (37.06%) C (10.33%)
adjacent nucs total GG (9.863%) UU (4.093%)
in helix GG (14.06%) AA (1.981%)
3'helix 5'loop CG (14.75%) UC(1.495%)
loop/helix ratio AA (5.67934) CC (.112825)
base pairs total GC/CG (28.29%) CU/UC (0.1351%)
5'helix end GC (38.76%) UC (0.09088%)
3'helix end CG (38.77%) CU (0.09089%)
Highest
three nucs total GGG (3.0%), GAA
(2.6%), AAG (2.6%),
GGA (2.5%), AGG
(2.4%)
000 GAA (7.5%), AAA
(6.7%), UAA (5.2%)
011 AGC (9.3%), AGG
(8.8%)
100 CGA (7.6%), UGA
(5.8%), GGA (5.3%)
110 GCG (6.9%), GGG
(4.8%), GGA (4.7%)
001 AAG (14.4%), AAC
(6.9%), GAG (5.4%)
101 CAG (7.2%)

Page 28 of 31
alignment editor AE2 (T. Macke, Scripps Clinic, San Di-
ego, CA). The rRNA alignments are sorted by phylogeny
and cell location, the intron alignments are sorted by sub-
group, exon, insertion point (for rRNA introns), and phy-
logeny, 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 re-
ferred to in rRNA GenBank entries without the intron se-
quence being provided; in these cases, we represent the
intron as having length "NSEQ" (No SEQuence informa-
tion) 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 classifi-
cations "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 clas-
sified as "Unknown" in the "RNA Class" field (see Section
4A and Table 4); included in this category are the Archaeal
and spliceosomal introns. At present, the Archaeal and
spliceosomal introns are identified with the phrases "Ar-
chaeal" and "spliceosomal," respectively, in the Comment
field of the RDBMS; a standard designation for these in-
trons will be added to a future version of the system. Al-
though 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 as-
sure 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 anno-
tated); and 3) Chara sp. Qiu 96222 (AF191800; the intron
annotations were shifted approximately 15 positions to-
ward 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 se-
quence length and percent completeness are instead rep-
resented 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.edu] described 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 phyl-
ogeny; 3) cell location; 4) RNA type (general category; e.g.,
rRNA or intron); 5) RNA class (more detailed identifica-
tion; 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 in-
trons 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 au-
tomatically during the incorporation process and then up-
dated 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 de-
veloped 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; [ht-
tp://www.cs.wisc.edu/~ghost/index.htm] ).
Computer details
Hardware and software used
The Comparative RNA Web Site [http://www.rna.ic-
mb.utexas.edu] is hosted on a Sun Microsystems Enter-
prise 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

Page 29 of 31
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 author-
ization system for its users. Information is collected to as-
sist 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 re-
view the terms and conditions for use of the CRW Site. An
email will be sent to the provided email address contain-
ing 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 pass-
word 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.edu] using
its main address, [http://www.rna.icmb.utexas.edu/] ,
rather than through specific URLs. As the site grows, spe-
cific 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 sys-
tem is configured to route an initial request for a more
specific URL to an introductory page, which will offer us-
ers 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
Acknowledgements
This work was supported by the National Institutes of Health (GM48207),
the Welch Foundation (F-1427), startup funds from the Institute for Cellu-
lar and Molecular Biology at the University of Texas at Austin (awarded to
RRG), and funding from the Ibis Therapeutics division of Isis Pharmaceuti-
cals. J. Collett was supported from NSF IGERT grant DGE-0114387.
We thank John Eargle, Daniella Konings Viloya Schweiker, Chris Simmons,
Bryn Weiser, and Ping Ye for their contributions to this project.
References
1. Darwin, C: Origin of Species by Means of Natural Selection, or
the Preservation of Favored Races in the Struggle for Life.
First edition, 1859; second edition, 1860; third edition, 1861; fourth edition,
1866; fifth edition, 1869; sixth and final edition, 1872. Amherst NY, Pro-
metheus Books.
2. Woese CR, Fox GE: Phylogenetic structure of the prokaryotic
domain: the primary kingdoms. Proc Natl Acad Sci USA. 1977,
74:5088-5090
3. Woese CR, Magrum LJ, Fox GE: Archaebacteria. J Mol Evol 1978,
11:245-251
4. Woese CR: Bacterial evolution. Microbiol Rev. 1987, 51:221-271
5. Holley RW, Apgar J, Everett GA, Madison JT, Marquisee M, Merrill
SH, Penswick JR, Zamir A: Structure of a ribonucleic acid. Science
1965, 147:1462-1465
6. RajBhandary UL, Stuart A, Faulkner RD, Chang SH, Khorana HG: Nu-
cleotide sequence studies on yeast phenylalanine sRNA. Cold
Spring Harb Symp Quant Biol 1966, 31:425-434
7. Madison JT, Everett GA, Kung HK: On the nucleotide sequence
of yeast tyrosine transfer RNA. Cold Spring Harb Symp Quant Biol
1966, 31:409-416
8. Zachau HG, Dutting D, Feldman H, Melchers F, Karau W: Serine
specific transfer ribonucleic acids. XIV. Comparison of nu-
cleotide sequences and secondary structure models. Cold
Spring Harb Symp Quant Biol 1966, 31:417-424
9. Levitt M: Detailed molecular model for transfer ribonucleic
acid. Nature 1969, 224:759-763
10. Kim SH, Suddath FL, Quigley GJ, McPherson A, Sussman JL, Wang
AH, Seeman NC, Rich A: Three-dimensional tertiary structure
of yeast phenylalanine transfer RNA. Science 1974, 185:435-440
11. Robertus JD, Ladner JE, Finch JT, Rhodes D, Brown RS, Clark BF, Klug
A: Structure of yeast phenylalanine tRNA at 3Å resolution.
Nature 1974, 250:546-551
12. Fox GE, Woese CR: 5S RNA secondary structure. Nature 1975,
256:505-507
13. Fox GE, Woese CR: The architecture of 5S rRNA and its rela-
tion to function. J Mol Evol 1975, 6:61-76
14. Brosius J, Palmer ML, Kennedy PJ, Noller HF: Complete nucleotide
sequence of a 16S ribosomal RNA gene from Escherichia co-
li. Proc Natl Acad Sci USA. 1978, 75:4801-4805
15. Woese CR, Magrum LJ, Gupta R, Siegel RB, Stahl DA, Kop J, Craw-
ford N, Brosius J, Gutell R, Hogan JJ, Noller HF: Secondary struc-
ture model for bacterial 16S ribosomal RNA: phylogenetic,
enzymatic and chemical evidence. Nucl Acids Res 1980, 8:2275-
2293
16. Zwieb C, Glotz C, Brimacombe R: Secondary structure compar-
isons between small subunit ribosomal RNA molecules from
six different species. Nucl Acids Res 1981, 9:3621-3640
17. Stiegler P, Carbon P, Zuker M, Ebel JP, Ehresmann C: [Secondary
and topographic structure of ribosomal RNA 16S of Es-
cherichia coli]. C R Seances Acad Sci D. 1980, 291:937-940
18. Brosius J, Dull TJ, Noller HF: Complete nucleotide sequence of
a 23S ribosomal RNA gene from Escherichia coli. Proc Natl
Acad Sci USA. 1980, 77:201-204
19. Noller HF, Kop J, Wheaton V, Brosius J, Gutell RR, Kopylov AM, Do-
hme F, Herr W, Stahl DA, Gupta R, Woese CR: Secondary struc-
ture model for 23S ribosomal RNA. Nucl Acids Res 1981, 9:6167-
6189
20. Glotz C, Zwieb C, Brimacombe R, Edwards K, Kossel H: Secondary
structure of the large subunit ribosomal RNA from Es-
cherichia coli, Zea mays chloroplast, and human and mouse
mitochondrial ribosomes. Nucl Acids Res 1981, 9:3287-3306
21. Branlant C, Krol A, Machatt MA, Pouyet J, Ebel JP, Edwards K, Kossel
H: Primary and secondary structures of Escherichia coli MRE
600 23S ribosomal RNA. Comparison with models of sec-
ondary structure for maize chloroplast 23S rRNA and for
large portions of mouse and human 16S mitochondrial rR-
NAs. Nucl Acids Res 1981, 9:4303-4324
22. Noller HF, Woese CR: Secondary Structure of 16S Ribosomal
RNA. Science 1981, 212:403-411
23. Woese CR, Gutell R, Gupta R, Noller HF: Detailed analysis of the
higher-order structure of 16S-like ribosomal ribonucleic ac-
ids. Microbiol Rev 1983, 47:621-669
24. Noller HF: Structure of ribosomal RNA. Annu Rev Biochem 1984,
53:119-162
25. Haselman T, Camp DG, Fox GE: Phylogenetic evidence for ter-
tiary interactions in 16S-like ribosomal RNA. Nucl Acids Res
1989, 17:2215-2221

Page 30 of 31
26. Haselman T, Gutell RR, Jurka J, Fox GE: Additional Watson-Crick
interactions suggest a structural core in large subunit ribos-
omal RNA. J Biomol Struct Dyn 1989, 7:181-186
27. Larsen N: Higher order interactions in 23s rRNA. Proc Natl Acad
Sci U S A 1992, 89:5044-5048
28. Gutell RR, Larsen N, Woese CR: Lessons from an evolving rR-
NA: 16S and 23S rRNA structures from a comparative per-
spective. Microbiol Rev. 1994, 58:10-26
29. Gutell RR: Comparative sequence analysis and the structure
of 16 S and 23 S rRNA. In: Ribosomal RNA: Structure, Evolution,
Processing, and Function in Protein Biosynthesis 1996, 111-128
30. Michel F, Jacquier A, Dujon B: Comparison of fungal mitochon-
drial introns reveals extensive homologies in RNA secondary
structure. Biochimie 1982, 64:867-881
31. Cech TR: Conserved sequences and structures of group I in-
trons: building an active site for RNA catalysis-a review. Gene
1988, 73:259-271
32. Michel F, Westhof E: Modelling of the Three-dimensional Ar-
chitecture of Group I Catalytic Introns Based on Compara-
tive Sequence Analysis. J Mol Biol 1990, 216:585-610
33. Damberger SH, Gutell RR: A comparative database of group I
intron structures. Nucl Acids Res 1994, 22:3508-3510
34. Michel F, Umesono K, Ozeki H: Comparative and functional
anatomy of group II catalytic introns – a review. Gene 1989,
82:5-30
35. Yu N: Comparative Sequence Analysis of Group II Intron and
tmRNA and Database. M.A. thesis, University of Texas at Austin, 2000
36. James BD, Olsen GJ, Liu JS, Pace NR: The secondary structure of
ribonuclease P RNA, the catalytic element of a ribonucleo-
protein enzyme. Cell 1988, 52:19-26
37. Brown JW, Haas ES, James BD, Hunt DA, Liu JS, Pace NR: Phyloge-
netic analysis and evolution of RNase P RNA in proteobacte-
ria. J Bacteriol 1991, 173:3855-3863
38. Harris JK, Haas ES, Williams D, Frank DN, Brown JW: New insight
into RNase P RNA structure from comparative analysis of
the archaeal RNA. RNA 2001, 7:220-232
39. Romero DP, Blackburn EH: A conserved secondary structure
for telomerase RNA. Cell 1991, 67:343-353
40. Chen JL, Blasco MA, Greider CW: Secondary structure of verte-
brate telomerase RNA. Cell 2000, 100:503-514
41. Williams KP, Bartel DP: Phylogenetic analysis of tmRNA sec-
ondary structure. RNA 1996, 2:1306-1310
42. Guthrie C, Patterson B: Spliceosomal snRNAs. Annu Rev Genet
1988, 22:387-419
43. Zwieb C: Structure and function of signal recognition particle
RNA. Prog Nucleic Acid Res Mol Biol 1989, 37:207-234
44. Wimberly BT, Brodersen DE, Clemons WM Jr, Morgan-Warren RJ,
Carter AP, Vonhein C, Hartsch T, Ramakrishnan V: Structure of
the 30S ribosomal subunit. Nature 2000, 407:327-339
45. Ban N, Nissen P, Hansen J, Moore PB, Steitz TA: The complete
atomic structure of the large ribosomal subunit at 2.4 A res-
olution. Science 2000, 289:905-920
46. Gutell RR: Collection of Small Subunit (16S- and 16S-like) ri-
bosomal RNA structures. Nucl Acids Res 1993, 21:3051-3054
47. Gutell RR: Collection of Small Subunit (16S- and 16S-like) ri-
bosomal RNA structures: 1994. Nucl Acids Res 1994, 22:3502-
3507
48. Gutell RR, Fox GE: A compilation of large subunit RNA se-
quences presented in a structural format. Nucl Acids Res 1988,
16 Suppl:rl75-r269
49. Gutell RR, Schnare MN, Gray MW: A compilation of large subu-
nit (23S-like) ribosomal RNA sequences presented in a sec-
ondary structure format. Nucl Acids Res 1990, 18 Suppl:2319-
2330
50. Gutell RR, Schnare MN, Gray MW: A compilation of large subu-
nit (23S- and 23S-like) ribosomal RNA structures. Nucl Acids
Res 1992, 20 Suppl:2095-2109
51. Gutell RR, Gray MW, Schnare MN: A compilation of large subu-
nit (23S- and 23S-like) ribosomal RNA structures: 1993. Nucl
Acids Res 1993, 21:3055-3074
52. Schnare MN, Damberger SH, Gray MW, Gutell RR: Comprehen-
sive Comparison of Structural Characteristics in Eukaryotic
Cytoplasmic Large Subunit (23S-like) Ribosomal RNA. J Mol
Biol 1996, 256:701-719
53. Olsen GJ, Overbeek R, Larsen N, Marsh TL, McCaughey MJ, Maciuke-
nas MA, Kuan WM, Macke TJ, Xing Y, Woese CR: The Ribosomal
Database Project. Nucl Acids Res 1992, 20 Suppl:2199-2200
54. Maidak BL, Cole JR, Lilbum TG, Parker CT Jr, Saxman PR, Farris RJ,
Garrity GM, Olsen GJ, Schmidt TM, Tiedje JM: The RDP-II (Ribos-
omal Database Project). Nucl Acids Res 2001, 29:173-174
55. Erdmann VA, Huysmans E, Vandenberghe A, De Wachter R: Collec-
tion of published 5S and 5.8S ribosomal RNA sequences. Nucl
Acids Res 1983, 11:rl05-rl33
56. Huysmans E, De Wachter R: Compilation of small ribosomal
subunit RNA sequences. Nucleic Acids Res. 1986, 14 Suppl:r73-
118
57. Van de Peer Y, De Rijk P, Wuyts J, Winkelmans T, De Wachter R:
The European small subunit ribosomal RNA database. Nucl
Acids Res 2000, 28:175-176
58. De Rijk P, Van de Peer Y, Chapelle S, De Wachter R: Database on
the structure of large ribosomal subunit RNA. Nucl Acids Res
1994, 22:3495-3501
59. Wuyts J, De Rijk P, Van de Peer Y, Winkelmans T, De Wachter R:
The European Large Subunit Ribosomal RNA Database. Nucl
Acids Res 2001, 29:175-177
60. Benson DA, Karsch-Mizrachi I, Lipman DJ, Ostell J, Rapp BA, Wheeler
DL: GenBank. Nucl Acids Res 2000, 28:15-18
61. Wheeler DL, Chappey C, Lash AE, Leipe DD, Madden TL, Schuler
GD, Tatusova TA, Rapp BA: Database resources of the National
Center for Biotechnology Information. Nucl Acids Res 2000,
28:10-14
62. Gutell RR, Cannone JJ, Konings D, Gautheret D: Predicting U-
turns in Ribosomal RNA with Comparative Sequence Anal-
ysis. J Mol Biol 2000, 300:791-803
63. Gutell RR, Cannone JJ, Shang Z, Du Y, Serra M: A Story: Unpaired
Adenosines in Ribosomal RNAs. J Mol Biol 2000, 304:335-354
64. Elgavish T, Cannone JJ, Lee JC, Harvey SC, Gutell RR: AA.AG@He-
lix.Ends: A:A and A:G Base-pairs at the Ends of 16 S and 23
S rRNA Helices. J Mol Biol 2001, 310:735-753
65. Konings DAM, Gutell RR: A comparison of thermodynamic
foldings with comparatively derived structures of l6S and
16S-like rRNAs. RNA 1995, 1:559-574
66. Fields DS, Gutell RR: An Analysis of Large rRNA Sequences
Folded by a Thermodynamic Method. FoldDes 1996, 1:419-430
67. Lydeard C, Holznagel WE, Schnare MN, Gutell RR: Phylogenetic
Analysis of Molluscan Mitochondrial LSU rDNA Sequences
and Secondary Structures. Mol Phylogenet Evol 2000, 15:83-102
68. Vernon D, Gutell RR, Cannone JJ, Rumpf RW, Birky CW Jr: Accel-
erated Evolution of Functional Plastid rRNA and Elongation
Factor Genes Due to Reduced Protein Synthetic Load After
the Loss of Photosynthesis in the Chlorophyte Alga Polyto-
ma. Mol Biol Evol 2001, 18:1810-1822
69. Bhattacharya D, Cannone JJ, Gutell RR: Group I Intron Lateral
Transfer Between Red and Brown Algal Ribosomal RNA.
Curr Genet 2001, 40:82-90
70. Gutell RR, Weiser B, Woese CR, Noller HF: Comparative Anato-
my of 16-S-like Ribosomal RNA. Prog Nucleic Acid Res Mol Biol
1985, 32:155-216
71. Gutell RR, Power A, Hertz GZ, Putz EJ, Stormo GD: Identifying
constraints on the higher-order structure of RNA: continued
development and application of comparative sequence anal-
ysis methods. Nucl Acids Res 1992, 20:5785-5795
72. Gautheret D, Damberger SH, Gutell RR: Identification of base-tri-
ples in RNA using comparative sequence analysis. J Mol Biol
1995, 248:27-43
73. Olsen GJ: Comparative analysis of nucleotide sequence data.
Ph.D. thesis, University of Colorado Health Sciences Center, 1983
74. Chiu DK, Kolodziejczak T: Inferring consensus structure from
nucleic acid sequences. Comput Appl Biosci 1991, 7:347-352
75. Correll CC, Freeborn B, Moore PB, Steitz TA: Metals, motifs, and
recognition in the crystal structure of a 5S rRNA domain. Cell
1997, 91:705-712
76. Cate JH, Gooding AR, Podell E, Zhou K, Golden BL, Kundrot CE,
Cech TR, Doudna JA: Crystal structure of a group I ribozyme
domain: principles of RNA packing. Science 1996, 273:1678-
1685
77. Hill WE, Dahlberg AE, Garrett RA, Moore PB, Schlessinger D, Warn-
er JR, editors: The Ribosome: Structure, Function, and Evolu-
tion. Washington DC, American Society for Microbiology 1990

Page 31 of 31
78. Zimmerman RA, Dahlberg AE, editors: Ribosomal RNA: Struc-
ture, Evolution, Processing, and Function in Protein Biosyn-
thesis. BocaRaton, CRC Press 1996
79. Neefs JM, Van de Peer Y, Hendriks L, De Wachter R: Compilation
of small ribosomal subunit RNA sequences. Nucl Acids Res.
1990, 18 Suppl:2237-2317
80. Woese CR, Kandler O, Wheelis ML: Towards a natural system of
organisms: proposal for the domains Archaea, Bacteria, and
Eucarya. Proc Nati Acad Sci USA 1990, 87:4576-4579
81. Sprinzl M, Dank N, Nock S, Schon A: Compilation of tRNA se-
quences and sequences of tRNA genes. Nucl Acids Res 1991, 19
Suppl:2127-2171
82. Suh SO, Jones KG, Blackwell M: A Group I Intron in the Nuclear
Small Subunit rRNA Gene of Cryptendoxyla hypophloia, an
Ascomycetous Fungus: Evidence for a New Major Class of
Group I Introns. J Mol Evol 1999, 48:493-500
83. Kjems J, Garrett RA: Ribosomal RNA introns in archaea and ev-
idence for RNA conformational changes associated with
splicing. Proc Natl Acad Sci U S A 1991, 88:439-443
84. Bhattacharya D, Lutzoni F, Reeb V, Simon D, Nason J, Fernandez F:
Widespread occurrence of spliceosomal introns in the rDNA
genes of ascomycetes. Mol Biol Evol 2000, 17:1971-1984
85. Maidak BL, Olsen GJ, Larsen N, Overbeek R, McCaughey MJ, Woese
CR: The RDP (Ribosomal Database Project). Nucl Acids Res
1997, 25:109-111
86. Van Oppen MJH, Olsen JL, Stam WT: Evidence for Independent
Acquisition of Group I Introns in Green Algae. Mol Biol Evol
1993, 10:1317-1326
87. Altschul SF, Gish W, Miller W, Myers EW, Lipman DJ: Basic local
alignment search tool. J Mol Biol. 1990, 215:403-410
88. Lutzoni F, Pagel M, Reeb V: Major fungal lineages are derived
from lichen symbiotic ancestors. Nature 2001, 411:937-940
Publish with BioMed Central and every
scientist can read your work free of charge
"BioMedcentral will be the most significant development for
disseminating the results of biomedical research in our lifetime."
Paul Nurse, Director-General, Imperial Cancer Research Fund
Publish with BMC and your research papers will be:
available free of charge to the entire biomedical community
peer reviewed and published immediately upon acceptance
cited in PubMed and archived on PubMed Central
yours - you keep the copyright
editorial@biomedcentral.com
Submit your manuscript here:
http://www.biomedcentral.com/manuscript/
BioMedcentral.com

Gutell 080.bmc.bioinformatics.2002.3.2

More Related Content

What's hot (7)

Viewers also liked (17)

Similar to Gutell 080.bmc.bioinformatics.2002.3.2 (20)

More from Robin Gutell (20)

Recently uploaded (20)

Gutell 080.bmc.bioinformatics.2002.3.2