1. 3502 -3507 Nucleic Acids Research, 1994, Vol. 22, No. 17
Collection of small subunit (16S- and 16S-like) ribosomal
RNA structures: 1994
Robin R.Gutell
MCB Biology, Campus Box 347, University of Colorado, Boulder, CO 80309-0347, USA
ABSTRACT
A collection of diverse 16S and 1 6S-like rRNA
secondary structure diagrams are available. This set
of rRNAs contains representative structures from all of
the major phylogenetic groupings Archaea,
(eu)Bacteria, and the nucleus, mitochondrion, and
chloroplast of Eucarya. Within this broad phylogenetic
sampling are examples of the major forms of structural
diversity currently known for this class of rRNAs. These
structure diagrams are available online through our
computer-network WWW server and anonymous ftp, as
well as from the author in hardcopy format.
INTRODUCTION
Comparative structure analysis of 16S rRNA has progressed for
the past 15 years. Initially with a minimal number of sequences
and a simpler perception of comparative analysis, a secondary
structure common to the few 16S and 16S-like rRNA sequences
was proposed (reviewed in 1,2.). Since then, the number of
publicly available and complete (or nearly complete) 16S and
16S-like rRNA sequences has increased most dramatically. As
ofJune 1994, this number is approximately 3,100 (see Table 1;
Ribosomal RNA Database Project see article in this issue;
RRG unpublished collection). In parallel with this pronounced
increase in 16S rRNA sequences, our comparative methods and
interpretation have evolved, resulting in a more detailed 16S
rRNA secondary and tertiary structure (reviewed in 1).
The details of the current secondary and tertiary structure for
16S rRNA should not be considered final. While the vast majority
ofthe proposed secondary and tertiary structure interactions are
considered correct given their high degree of comparative
evidence, alterations in these structural elements are not
anticipated. However, a small number of proposed interactions
are considered tentative, for these contain a minimal amount of
comparative evidence and are thus candidates for secondary
structure adjustments in the future. Further refinement in 16S
rRNA higher-order structure will also result from the analysis
of a larger number of aligned sequences with improved
correlation analysis methods (3; Gautheret, Damberger, and
Gutell, mss. in preparation). These latter structural constraints
are likely to involve base triples, non-canonical pairings, and
other structural elements more complex than simple secondary
structure base pairings.
OBJECTIVES
The primary goals for this 16S rRNA database are to: (1) Present
the most recent comparatively inferred secondary and tertiary
structure for 16S rRNA. Minor refinements in these structures
are expected, as we identify new (or alternative) secondary and
tertiary interactions. (2) Offer a sampling of representative 16S
and 16S-like secondary structure diagrams. The initial selection
of structures are phylogenetically as well as structurally distinct
from one another (see Table 2). Over time additional examples
ofdiverse structures will be generated, as well as structures that
are closely related to members of this database. (3) Three
divergent 16S-like rRNA diagrams are presented here. They are:
Haloferax volcanii, a representative Archaea (5, Figure 1);
Phreatamoeba balamuthi, a lower Eucarya that contains several
large insertions (6, Figure 2); and the Chiamvydomonas reinhardtii
mitochondrion; a most bizarre rRNA that is coded for by a
fragmented rRNA gene (7, Figure 3).
DATABASE
The higher-order structures described herein will be available
on-line or in hardcopy format from the author. On-line access
is by anonymous ftp or through the WWW (World Wide Web).
These computer files will be distributed in PostScript format only.
Our ftp address and directory are:
pundit. colorado. edu (128.138.212.53)
/pub/RNA/16S
Our WWW server can be accessed by programs like XMOSAIC
(see Damberger and Gutell, this issue), that navigate the WWW.
With this system the secondary structures can be viewed and
Table 1. Phylogenetic distribution of publicly available 16S and 16S-like rRNA
sequences (as of June 1994)
Organism type # 16S rRNA
Archaea 120
(Eu)Bacteria 2130
Eucarya (nuclear) 700
Organelles
Mitochondrion 110
Plastid 40
TOTAL 3100
1994 Oxford University Press

2. Nucleic Acids Research, 1994, Vol. 22, No. 17 3503
Table 2. List of currently available 16S and 16S-like rRNA secondary structure diagrams. Phylogenetic classification is based on Woese
et al. (4)
Archaea
Euryarchaeota Archaeoglobus fulgidus, Halobacterium marismortui (HC8),
Halobacterium marismortui (HCIO), Haloferax volcanii,
Methanobacterium formicicum,
Methanospirillum hungatei, Methanococcus vannielli,
Thennococcus celer,
Thernoplasma acidophilum
Crenarchaeota Pyrodictium occultum, Sulfolobus solfataricus, Thermoproteus
tenax
(eu)Bacteria
Aquifecales Aquifex pyrophilus
Thermotoga Thermotoga maritima, Geotoga subterranea, Petrotoga miotherma
Deinococcus and relatives Thernus thennophilus
Spirochetes and relatives Borrelia burgdorferi, Leptonemna illini, Spirochaeta aurantia
Planctomyces and relatives Planctomyces staleyi, Gemmata obscuriglobus-1, Gemniata
obscuriglobus-2
Green sulfur bacteria Chlorobium vibrioforme
Green non-sulfur bacter. Thermomicrobium roseum
Cytophaga-Flexi.-Bacter. Bacteroides fragilis
Purple bacteria Agrobacterium tumefaciens, Pseudomonas testosteroni,
Escherichia coli, Dichelobacter nodosus, Myxococcus xanthus,
Desulfovibrio desulfuricans, Campylobacter sputorum subsp.
sputorum
Cyanobacteria Synechococcus sp. 6301
Gram-positive Heliobacterium chlorum, Arthrobacter globiformis, Frankia sp.,
Streptomyces coelicolor, Bacillus subtilis, Epulopiscium sp. (strain
Al), Clostriiwum innocuum, Mycoplasma hyopneumoniae,
Mycoplasma gallisepticum, Mycoplasma capricolum,
Eucarya (nuclear)
Archezoa Hexamita, Giardia muris, Giardia ardeae, Giardia intestinalis,
Encephalitozoon cunuculi, Vairimorpha necatrix
Protoctista Phreatamoeba balamuthi, Babesia bigemina, Chlorella luteoviridis,
Staurastrun - M752, Genicularia spirotaenia, Gracilariopsis sp.,
Tritrichomonas foetus
Fungi Saccharomnyces cerevisiae, Cryptococcus neoformans
Plantae Fragaria ananassa (strawberry)
Animalia Drosophila melanogaster, Placopecten magellanicus, Xenopus
laevis, Mus musculus, Homo sapiens
Mitochondrion
Protoctista Chlamnydomonas reinhardtii, Paramecium tetraulia, Tetrahymena
pyriformis, Physarum polycephalum
Fungi Saccharomnyces cerevisiae, Aspergillus nidulans
Plantae Zea mays (maize)
Animalia Bos taurus (bovine), Ascaris suum, Caenorhabditis elegans
Chloroplast
Protoctista Chlamydomonas reinhardtii, Chlorella vulgaris, Cryptomonas sp., Olisthodiscus luteus,
Astasia longa, Euglena gracilis, Pylaiella littoralis, Cyanidium caldarium, Palmaria palmata
Plantae Nicotiana tabacum (tobacco), Zea mays (maize), Marchantia polymorpha (liverwort)
downloaded, while ftp access only supports downloading of the
PostScript files. Our WWW URL address is:
URL:http://pundit. colorado. edu:8080/RNA/16S/16s. html
The work presented here complements the effort of the RDP
(Ribosomal Database Project, see this issue); Their objectives
are to generate and present various types of ribosomal RNA data
and interpretation. On-line access to the 16S rRNA secondary
structure PostScript files is also available from the RDP
anonymous ftp site (rdp.life.uiuc.edu). Additional information
about this server and access to the Ribosomal Database Project
is described in this special Database issue.
The interactive graphics program XRNA (developed by Bryn
Weiser, UC Santa Cruz) facilitated the generation ofthe higher-
order structure diagrams. Manual alignment of these 16S and
16S-like rRNA sequences was facilitated with the interactive
sequence alignment editor AE2 (developed by Tom Macke,
Scripps Clinic).

3. 3504 Nucleic Acids Research, 1994, Vol. 22, No. 17
U
G
U
GU AA C-G
C U ccC-GC AA
AAUCC CCGAGGACA GAUGGCG u A G C u GAG
A CCGGC AC
A G ACGAGC CCGCA CUUCUAA UGC CAGCAGUUU
UGAGGGGGCCUGCUGGA0 CGAA
G
111111 III 1111111 1.1 I II I I I I
AUG GGCG C AGG. ACG CS0A UGCUCG GGGCAA GAAGAUU AUG GUCGUCAG
AUGAG-C ~~~ ~~~~~~~G-CA C G G AC G G
G-C G-C C A G A
A-U A G U -A A C
G-C ~~ ~~~~~~~GUG-C A U
U -A AGAU AUGA C -G G G
C-G U A C-~~~~~~~~~~~~cG U GA GGGo A A C G c CA G
A A ~~~~A / UUC-G G'$
A oG G-C UGG AU GGC G
G A c-AC / CG GUGA AAU
G-C C -G /CAGAC G C-A G AG ACG A -UG A
C~~~GA~~AG C
A-U G A A u
C
-GUG-C UC-G G GUC
GG-C UA Gu G-C
u G ~~UG'U AGUGA /,UAG AG-C
AACUGGCG UCCGGUGAA CCACGUGGC A G-C U
U' G-CGA-U AcG cA G
C IIIIII111 *II I I II II I II 111 C
/GG U-AGC GN A G A
UCGACCGC GGGCUACUU GG GCACCG G //GUGA CGUG AGC,GA C C-GUC A AA Cc a AG CUGUA AcA UCc UAUAA G GG A A %G CGAu
AAA UG C
AGCC //,CG A UGA%GU UCUC G
Ua
C u-G
A ACC
uA AG %GU c GG
G GCUCGCUA C A C A
GCC
A
C GGCA cA G~ UU
AUCU%A
cm GGC-a GA G CG CaU AAU GC UUG
C A
a-CG G- A
A GC
G- ~~~G/G A A G C
AAUGG
C AA a-c CUC U. CACG UA U G AC ACUCUA
U A AU GGAAUCCCG C-G AG C
AGG a-C U A UUCC G-C GA UA C G~~-C U A11 A-U CUCAAAA G ~~C-a A UUCA UAGGAA G ACAAUGC
UGC/'lG AGAC-G U
AA
AAG AG UG G
CC A
~~~~~~~~~~~~~~UACACG-~GAGGCCa CUA
CA /C :C AGG G A~Cc ,u G
GCU0AG AC A GIIUGGGGGGGCi*'1AC1 A A
GAACGC CU C'G CAGCA U
ACUUAC UACaAA G C G U
U CUUGCUCG AU AC CUCUCG UGaA UNII _UA U UG UAA;AU_I A AUl CCUCA C77G A.A
CAG UCuG A CGUaG a A G*U
uAGUCCUCGCAG u A C / UC GU G.
CCU111 A 5U' C-U G A
u C A T~~~~~CAaAl AGUAAG Ga/A U~
AAGUGCGUAAG GG -C UGCCUGG a/ U
CAAG A ACCCu
CACU A A AaU GAUA G U
AC0G CG~~~~~UAC CA G-
AGG G-C~~~~~~UAGC UGG U A
CGGGUAG U UCCCCAA.A ~~~C.A a A A/ A c-C CG Ca- GAGU GC CA C
GCCGU uc
A- A A GAUUGGC AC U
A A CG
a-CG-C
C/GCG GU A C
CC// C U
CAAG a ~~~~~G: A a a
A CUjG GCU CGa GC -G GUACAGU 3'
A UU''' /GGGC GCCA C
AAG CGG UGCUCAAC
GUAIC AUUC A-U 3UU,CCGGC GAA A CUUACGGUUG.
G
AGGc/ CUUCUA UG
GCGC A AA A a A
G /C A C
CU/ CG A %C A U-
A GG C-G U-A
GCUACGGGUU
U.G-a
AAC CA C-a aCCa *UG a-Ca-C ACG
C C
U
GU C G
C C a-C C
a ACC-GAC aG: C
AA U A
-A
AAU~ ~ CA AU U
AGGC AGAGACCG
U A
UC8 CUUGA CGGAGUCA
A A UAAU
A U
Aa A
GC C
C-a
C A
C - U
a-C
U U
A AG a
FIgur 1. Higher-order structure diagram for Haloferax vokcanii 16S rRNA (5). Different typs of secondary structure, basepairings are distinquished as follows:
short lines connect canonical pairs (C:G, U:A), G:U pairs are denoted with dots, A:G pairings with larger open circles, and other non-canonical pairings with closed
circles. Terdary interactions are connected with thicker and longer solid lines. [Sequence Accession number is K00421].

4. Nucleic Acids Research, 1994, Vol. 22, No. 17 3505
AU GAA A
ACUG
AAUCC UACGGAAAGAUUA A GCG
G I *IIIIIIIII * II A
UGGGGA C GUGCCUUCAUAAU uA CGA
U U
G-C
AAUUCCAACACCCGGUUUCAAGUUGAAUGGAAAGAAGGGUAUAUUAGUAU Gz CU-A
UUAGRRGGAGCAAUCCUUUUAAGUAUUGRUGUAUCAGUUCACCGUUUAAC G-C
UCCGGGGAAGGCUAUCCACUCUAAUGCCUUAUUCCCGCCAGGGCUCUUAA G ° A
UUGAGUCUUGGAUAUACCACCCGGACCGAUAAGGAAUGGGGGGAAUAAGG G A
ACUAAAGGUUUACCUUGAACAAAUUAGAGUGAUUCAGGCAGGCUUUUUUA C-G
AGCCCUUGAAUACAUUAGCAUGGGACAAACACAUCCCAAAAACAACCUGG GC A
GUCGGGUGGCACCGGGGUUGGAGGUUUGAUAUGAGAGGAGAUUAUGAGUA A- U
GGUGGUUAUGAUCAUUUGUGCAAUCAAAACUUAUAGUAGCCUCAAUGGUU C-GU
CCGUUUGGUUUUCCUGAGGGGGCUCGGACUUGGUCAUCUGUAGGUAUUUC A- U
AGGGGUGAUGGGCUCCGGUCUGUUAUUCCUGAGGUAUUCGGAUGAUUGAG G. U
GGGUCUAACCAGCAAUGGUUAUAUUAUGAGUUUGUAUGAAUGAUUAU0AU -C
URCUCUURUAGUCGAYAAAGACUUCACGUGUCGGGCUUUCAGUCUUAGGA A
CCAUCCAUUAAGACUCAGUCGUUUUGGUUGUAGAAAUGCAAUCGAAACGU
IUAAACCAAAIUAAIA U
A
AAAC U
CA A U
GC AGU U
I I I II C
CG UCA U
G AA AU
a AU GGU
G G CA
CACCGACGGCCUGAUCGGGUGACCGAGAGGGUCGCACUUGUCUUAAUUCA
CAGUGCCCCGGAACUGAGGCUGUUCGACGUGGUAGGGGAGGACGCUGAAU
GGGGCUGGUAGAAACAACUGGGGGUAUAAAACCAAGGAGGAAGCAAAAAA
GCCAUAACCCGGCGAUGGCCUUGGUGGAAACCUCUG00CUCAAGGUUGUU
AUUAUGUUYAUURUGGCCUCUCGGGGUUAUUUURAAUGURGUAAUAAACC
GAAAGCAACUCUAUCAGUUUGGUUYGGAUGUCCGUUAAUCCUGCGUGGCC
AGCGGCUUUGGGACUCCAGGGGACAGGGCGAAACGAGGCAAUUCAAAGCU
GAU
U A
a _
UUUCYGUAGG U
I I -I I
G
GAAGACGUCCAAU
CA
A
A
G
CUUU
3,
I

5. 3506 Nucleic Acids Research, 1994, Vol. 22, No. 17
CUAGUGC CG GUCU
Cc CCA
C
AAU A C U
0 U ccOAGG U
AAUGAUCCGGAUGGAAAGGAAGCGA G C
A .1 III II*' * GU
C A GA c G
UGGCUAGGAGGCUUAUAAUG G-C A GAG A
G-C ~~~~~~~~~~~~~~~~~~~~AA
U-A G C
U-A C GUA
~~~~~~~~~~~~UU
G-C AGA U0 c
C A U G AG
AA U U A AAU
AU.-..AAAC0-C GUU .G 0-CAU -A C-G uc
C-G c cU-A
CA-U A A AU
AA C A GCO U
GA A 0 UU G
G U A~~~~~~AA
C5AAUA
A U-A U"AAUA
G_C~U- AUUUU
AA AA A A
GC/U cU UC A AUCAUU -A
CG0 A A
CACC-
A A Lcc uA U
A C 111111~ ~~~~CC0
G A
Ac U UUAII c e u A-
C0AA UG A
AUCu A17G0 A U- A AA U
UUCGUGG- 0GCAUC A-
GG A C ~~~~~-UGGUCAAC-G U .111G A 0AG- AC U UA
A- UA UCACA 0A UG C
C- A
C U AUA U U CAAAG- UA AC U* A
CCC-0CAAGU U ACACAAGAGAUGUCAAUAAC-GGAGOUC' UA
UU A- U CAAAU 0U CA~C A GU.cU0 C- G 0 A ACCUGUUC UGU C-G A GGC .
UGCUAUUGGAAUUUUACAAAC GC ACA
C0-UAC GA ,U A~G U AUC A 0
UGA-UA
u
u
GU c
A A_CGuACU AU A A G
3'-AAACAU 0CG A U
/IAU Gj u
UG
G
A 0- .C0 A0
G'C G
A
C AA G GAA GUG/UG A-
UGCu.0GAC A
c u: 0GC/A C_GAUGAc C G ~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~UUGGGGUAGGGAUC/ UC
ACGGCGGUCCAu u u 1~~~~.I1**~~ A A U
A 1I*- liii C GACCUCOUCCA 0 c
Gu~~~~~~~~CGCUAGGUUGG A~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~AA CGAU G
C u U C U 0 A c
AA ~A U C -
G U u C
U AGUG U -A C C A
U A C0 C C-G A A A
U0G U A 0 C- G A A A
U -A CC U G U A A
G G ~ ~~~~A A-UU o A
0-CA A U UGA U A-U A0U.0AUA A A C G0
UCGA AUCUACGC-GU A A -G U G
0 A0c A-U U A u
A
AGGG
G
~~A AA G Uz AU A
G ~~~~AAA CACGCUU 0 A-U C GA lii 11U U -A 3' U
AUAOGGUAAUAUU U0- A _A
A UACGC
UAA
AACAUAUUAUUUAAU 0 U U-A
AC IUUUI U G U C
A./ o 3' A C 0
U' AA AC G3CGCu-Acu.
GGCG c _U~~~~~C-C
G//cGAU GAUACA-U
UC / G G
A A
ACC0 A 0 UAA C A 0 GA.Uu A C U G
C C 0 A U U
C AU-AA A A Gc
UAU Go-GU AC-GG
U*ACC:
.GuGA-UC
UCC-C- G0-C
Au 0 A C C- G
A A A U *GC
CG A U A C
A CG
CC 0
u ~~~~
U C UAA
A G
Figure 3. Higher-order structure diagram for Chlamydomonas reinhardtii mitochondrial 16S-like rRNA (7). Higher-order structure interactions illustrated and noted
as in Fig. 1. [Sequence Accession number is X54860].
Figure 2. Higher-order structure diagram for the Eucarya, Phreatamoeba balamuthi nucleocytoplasmic 16S-like rRNA (6). Higher-order structure interactions illustrated
and noted as in Fig. 1. [Sequence acccession number is L23799].

6. Nucleic Acids Research, 1994, Vol. 22, No. 17 3507
Research benefiting from the secondary structure diagrams in
hardcopy format or from the online compendium should cite this
article.
ACKNOWLEDGEMENTS
I wish to thank Mr Simon Damberger for helping establish our
WWW server, the W.M.Keck Foundation for their generous
support of RNA science on the Boulder campus, and SUN
Microsystems for their timely donation of computer equipment.
The author is an Associate in the Program in Evolutionary
Biology of the Canadian Institute ofAdvanced Research. This
work was supported by the NIH (GM 48207).
REFERENCES
1. Gutell R.R., Larsen N., Woese C.R. (1994). Lessons from an Evolving
Ribosomal RNA: 16S and 23S rRNA Structure from a Comparative
Perspective. Microbiological Reviews. 58,10-26.
2. Gutell R.R. (1993). Comparative Studies of RNA: Inferring Higher-Order
Structure from Patterns of Sequence Variation. Current Opinion in Structunal
Biology. Section: Nucleic Acids. 3,313-322.
3. Gutell R.R., Power A., Hertz G.J., Putz E.J., and Stormo G.D. (1992).
Nucleic Acids Res. 20,5785-5795.
4. Woese C.R., Kandler O., and Wheelis M.L. (1990). Towards a natural system
of organisms: Proposal for the domains Archaea, Bacteria, and Eucarya.
Proc. Natl. Acad. Sci. USA 87,4576-4579.
5. Gupta,R., Lanter,J.M. and Woese,C.R. (1983). Sequence of the 16S
ribosomal RNA from Halobacteriwn volcanii, an archaebacterium. Science
221, 656-659.
6. Hinkle,G., Leipe,D.D., Nerad,T.A. and Sogin,M.L. (1994). The unusually
long small subunit ribosomal RNA ofPhreamamoeba balanuwhi. Nucleic Acids
Res. 22, 465-469.
7. Boer,P.H. and Gray,M.W. (1988). Scrambled ribosomal RNA gene pieces
in Chlamydomonas reinhardtii mitochondrial DNA. Cell 55, 399-411.