1. +
Chapter 6
Comparative Sequence Analysis and the Structure
of 16S and 23S rRNA
Robin R. Gutell
CONTENTS
+
I. Introduction .................................................................................................................................. III
A. General Comments ............................................................................................................... III
B. Principles of Covariance Analysis ........................................................................................ 112
C. Modelling rRNA Secondary and Tertiary Structure ............................................................ 112
II. Secondary and Tertiary Structure ............................................................................................... 115
A. Current Models of 16S and 23S rRNA Secondary and Tertiary Structure ........................ 115
B. Characteristics of the Base Pairs .......................................................................................... 117
1. Canonical Pairings ........................................................................................................... 117
2. Noncanonical Pairings ..................................................................................................... 118
a. G:U Base Pairs and G:U .... A:C Interchanges .......................................................... 118
b. Purine:Purine, Pyrirnidine:Pyrirnidine, and Other Interconversions .......................... 119
C. Artangement of the Base Pairs and Characteristics of Helices ........................................... 120
1. Traditional Organization of Base Pairs and Helices ....................................................... 120
2. Lone Pairs ........................................................................................................................ 121
3. Pseudoknots ..................................................................................................................... 121
4. Base Pairings in Parallel .................................................................................................. 122
5. Base Triples ..................................................................................................................... 122
6. Coaxial Helices ................................................................................................................ 123
7. Tetraloops ........................................................................................................................ 123
m. A Comparative Perspective on the Structure of rRNA .............................................................. 124
Acknowledgments ................................................................................................................................ 125
References .............................................................................................................................................. 125
I. INTRODUCTION
A. GENERAL COMMENTS
Determining the secondary and tertiary structure of an RNA from its sequence requires a strong
understanding of the basic principles of RNA structure and the expertise to utilize these principles to
transfonn the sequence of that molecule into its higher-order structure. Beyond our basic appreciation for
secondary-structure helices and a few emerging RNA structural motifs (e.g., pseudoknots, tetraloops.
etc.), our knowledge of the principles of RNA structure is rudimentary. Moreover, our ability to fold a
primary structure into its secondary structure. while getting better, needs further improvement since the
number of theoretically possible secondary structures for large RNA molecules is quite large, and
identifying the biologically correct version is not easily accomplished. Given these limitations, we are
unable to take any single rRNA sequence and detennine its secondary or tertiary structure with strong
confidence. However, the methodology of comparative sequence analysis has been used to solve the
secondary structures for a number of different RNA molecules (reviewed in: Woese and Pace 1993,
Gutell 1993a). The objective of this chapter is to review comparative sequence analysis and the structures
for 16S and 23S rRNA that have been inferred with this approach, and to discuss some of the principles
of RNA structure that.are resulting from these studies. [A longer and more detailed account ofthe material
presented in this article has been published elsewhere (Gutell, Larsen, and Woese 1994)].
Several different RNA molecules have been analyzed by the comparative approach. Comparative
sequence analysis was first used to suggest the cloverleaf secondary-structure configuration of tRNA
(Holley et al. 1965; Madison, Everett, and Kung 1966; RajBhandary et al. 1966; Zachau et al. 1966). A
0-S49J.Il864-JI96I$O.oo.s.so
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few years later a more detailed analysis yielded comparative support for afew tertiary interactions within
lRNA (Levitt 1969). Today, with a significantly larger collection of lRNA sequences, and more refined
and quantitative covariation methods. all of the secondary-structure base pairs and the majority of tertiary
base-base interactions (Olsen 1983; Gutell et al. 1992; Gutell 1993a - and references therein) and tertiary
base-triple interactions (Gautheret, Damberger, and Gutell 1995) can now be inferred.
B. PRINCIPLES OF COVARIANCE ANALYSIS
Comparative sequence analysis is based on a very simple and profound principle. The same three-
dimensional structure for an RNA molecule can be derived from a h~rge number of different sequences.
For this paradigm to be proven, the functionally equivalent RNA molecules under study (e.g., lRNAs, 16S
rRNA. etc.) should form a comparable three-dimensional structure no matter how similar or divergent
their sequences are. Trus principle has been applied mainly in the following circumstances. Analogous
secondary- and tertiary-structure helices have been proposed when compensatory base changes (e.g., A:U
H G:C) in a collection of aligned sequences are identified within a potential helix. In other words, (two)
positions are said to have a structural relationship (Le., base pair) when their patterns of change in a
sequence alignment are coordinated (i.e., the two positions covary). Thus, to identify secondary-structure
helices we search for positional covariances. The first and simplest result to emerge from this analysis
was the identification of secondary-structure helices. Subsequently, tertiary base-base interactions have
been proposed on the basis of these same methods. More recently, refined methods for identifying base-
triples (Gautheret, Damberger, and Gutell 1995) have been developed and applied to the rRNA datasets,
resulting in several likely base-triple candidates (Gutell et aI., mss. in preparation).
Comparative analysis reveals more than just the secondary- and tertiary-structure pairing assignments
between two or three bases. The pattern of variation between the two pairing positions can suggest a
specific confonnation for the pairing arrangement. A few examples are mentioned here. (I) Two positions
that contain all canonical and G:U base pairing possibilities are quite likely to fonn the standard Watson-
Crick base pairing confonnation. (2) A few base-paired positions in 16S and 23S rRNAs are restricted
to A:U or U:A base pairs, suggesting that these base pairs fonn a unique conformation that only this
combination of pairing types can adopt. (3) For some base pair positions. the constraint is different among
various phylogenetic groups, implying that different confonnations might occur within different phylo-
genetic groups. A sampling of other restricted base pair exchanges are discussed below.
The comparative approach by itself does not prove the existence of a base pair, helix, or entire
secondary structure. Instead it reveals constraints in positional variation, from which we infer secondary
and tertiary structure. Given the success this approach has had in predicting a three-dimensional tRNA
structure that is largely congruent with its crystal structure solution, we are confident that these inferred
structures are biologically meaningful. While the comparatively derived 16S and 23S rRNA secondary
and tertiary structures cannot be experimentally substantiated in the same manner as tRNA, the combi-
nation of various experimental approaches (e.g., site-directed mutagenesis, chemical modification, etc.)
has corroborated these proposed structures (see Hill et al. 1990; Nierhaus et al. 1993). The comparative
methodology should not be viewed as a competing approach in elucidating rRNA structure. Rather the
combination of experimental and comparative approaches presents us with a richer collection of facts to
build upon. The comparative approach is, in one sense, an analysis of experiments perfonned during the
evolution of the rRNAs under study. However, we do not know what the experimental design or intents
were. We observe and analyze the sequences that have survived this evolutionary process, and from these
patterns of variation we have inferred secondary- and tertiary-structure base pairings. We can also derive
other types of infonnation from these patterns of sequence variation. For example. positions and structural
features that are highly conserved are indicative of functional significance. The compositional frequency
within certain unpaired loops could suggest unique conformations or thennodynamic properties (e.g.,
"tetraloops"; see below).
C. MODELLING rRNA SECONDARY AND TERTIARY STRUCTURE
The ribosomal RNAs are quite amenable to the comparative approach due to their significant role in the
structure of the ribosome and its function in protein synthesis, their long biological ancestry that links
them to an early stage in the evolution of the cell, and their patterns of conservation that make them the
preferred choice for phylogenetic reconstruction studies (Woese 1980, Woese 1987).
The modelling of rRNA structures has proceeded in stages. The comparative analysis has relied
primarily on the simplest pattern of covariation. Two positions are considered paired only when the two

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aligned positions vary in a highly coordinated manner (i.e., variation at one position is compensated for
by a correlated change at its pairing position). Initially, only covarying positions with canonical (A:U and
G:C) and G:U pairings, flanked by other canonical pairings, were considered to be base paired. The initial
studies on 16S and 23S rRNAs resulted in minimal secondary-structure models (Woese et al. 1980;
Stiegler et al. 1981; Zwieb, Glotz, and Brimacombe 1981; Noller et al. 1981; Branlant et al. 1981; Glotz,
Zwieb, and Brimacombe 1981). The majority of the proposed helices are still present in our current
secondary- structure model, although many have been refined. At the outset, with a small number of
available sequences, only a few base pairs within a potential helix exhibited compensatory base substi-
tutions. In such cases, the length of the helix was determined to be the maximum number of possible
consecutive and canonical base pairs. At that time a helix was considered phylogenetically proven when
two or more of its base pairs had two or more compensatory base changes (Woese et al. 1980).
The growing rRNA sequence collections presented us with the opportunity to test the authenticity of
the first comparatively derived structures. and to search for new interactions. The majority of the earlier
secondary-structure base pairs in the 16S and 23S rRNAs were corroborated with the additional sequence
infonnation. These base pairings either received support from additional examples of compensatory base
substitutions, or the nucleotides that are putatively base paired were phylogenetically conserved in the
larger rRNA datasets (i.e., they did not contain counter-evidence). Thus the number of base pairs with
comparative support (i.e., compensatory base changes) increased with the larger number of available
sequences. In contrast, some base pairs were eliminated due to uncompensated base substitutions (Le.,
a change at one position without a change in its putative pairing partner). New positional covariances were
identified with these larger sequence collections. Some of these pairings were components of newly
proposed secondary-structure helices. Others were associated with "more complex secondary (e.g.,
noncanonical base pairings, or base pairings not flanked by other canonical base pairs; see below) or
tertiary (e.g., pseudoknots and parallel interactions; see below) interactions.
The first complete 16S rRNA sequence was determined in 1978 (Brosius et al. 1978, Brosius et al.
1981). Subsequently, approximately 3,750 16S and 16S-like rRNA sequences (complete or nearly so)
have been determined and are in the public domain (or will be soon) (Maidak et al. 1994, Gutell 1994,
Gutell, unpublished collections). This number includes 2,200 from (eu)Bacteria, 100 from Archaea, 1,220
from Eucarya (nuclear), 60 from chloroplasts, and 160 from mitochondria. In 1980, the first complete 23S
rRNA sequence was published (Brosius, Dull, and Noller 1980). Since then, approximately 340 23S and
23S-like rRNA sequences (complete or nearly so) have become publicly available (or will be soon)
(Gutell, Gray, and Schnare 1993; Gutell, unpublished collection). Ofthis number, 82 are from (eu)Bacteria,
16 from Archaea, 42 from Eucarya (nuclear), 92 from chloroplasts, and 105 from mitochondria. This
collection of sequences is large and well distributed across all of the major taxa. Primary-structure
alignments and secondary-structure diagrams for representative 16S and 23S rRNA sequences are also
publicly available (Maidak et al. 1994; Gutell 1994; Gutell, Gray, and Schnare 1993).
In parallel with the increase in the number of 16S and 23S rRNA sequences, the methods used to
identify positional covariances have improved. Initially, only covariations involving canonical base
pairs in a potential secondary structure helix were scored positively. Later on, a more sophisticated
algorithm was developed to identify positional covariations regardless of the base pair types and the
interactions at the flanking positions (Gutell et al. 1985). However, this algorithm could only identify
perfect covariations (e.g., A:U H G:C interchanges were identified, but A:U H G:U H G:C were
missed). Currently, our algorithms measure the mutual information between two positions with a chi-
square statistic (Gutell et al. 1992), and a pseudo-measure of the number of mutual changes that have
occurred during the phylogenetic evolution ofthe RNA under study (Le., phylogenetic events) (Gautheret,
Damberger, and Gutell 1995; Gutell and Damberger, mss. in preparation). These newest computational
methods are currently being applied to our very large collection of 16S and 23S rRNA databases. The
16S rRNA secondary and tertiary structure model remains largely the same, although a few secondary
structure base pairings will be eliminated, while a few tertiary-like pairings will be added. Several
putative base triples have been identified as well (Gutell et aI., mss. in preparation). For the 23S rRNA,
the refinements in structure will involve a few more base pairings. A few pairings have been eliminated
(which are reflected in Figure 1). while a greater number of new secondary and tertiary base pairs will
be included in future structure models. Several strong base triple candidates have now been identified
(these new base-base and base-triple interactions are not shown in Figure I). Of these newly proposed
23S rRNA interactions, several are located in functionally important regions of the 23S rRNA (Gutell
et al.. mss. in preparation).

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Figure 1A The current version of the Escherichia coli 168 and 238 rRNA secondary and tertiary structure
models. A detailed comparative analysis for the secondary and tertiary structure base pairs will be formally
presented elsewhere. All canonical (A:U, U:A, C:G, and G:C) secondary-structure base pairs are indicated by a
connecting line, G:U pairs by a dot. G:A pairs with open circles, and other noncanonicat pairings (see text) with
closed circles, The nucleotides that are juxtaposed but not connected with a line or circles are considered
possible, but do not have comparative proof or disproof. These nucleotides are usually invariant, or nearly so.
"Tertiary" interactions with strong comparative data are connected by thicker (and longer) solid lines. Dashed
tertiary lines refer to interactions that are considered possible, but are lacking convincing comparative evidence.
Every 10th nucleotide is marked with a tick mark, and every 50th nucleotide is numbered. The secondary structure
diagrams were drawn with the program XRNA, which was developed by B. Weiser and H. Noller. (A) -
Eschen'chia coli 165 rRNA. The sequence was determined by Brosius at aI. (Brosius et aJ. 1978; Brosius et aJ.
1981 ).

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Figure 1B Escherichia coli 238 rRNA, 5' half. The sequence was determined by Brosius et al. (Brosius et al.
1980.)
II. SECONDARY AND TERTIARY STRUCTURE
A. CURRENT MODELS OF 16S AND 23S rRNA SECONDARY AND TERTIARY
STRUCTURE
The comparatively inferred secondary- and tertiary-structure models for Escherichia coli l6S and 23S
rRNAs are shown in Figure I. The base numbering in Figure 1 and within this chapter are for the E. coli
l6S and 23S rRNA sequences (Brosius et al. 1978; Brosius, Dull, and Noller 1980; Brosius et al. 1981).
The degree of comparative support for the majority of the secondary and tertiary base pairs in the 16S
and 23S rRNA structure models is quite strong. A small number of pairings reflect minimal comparative
evidence, although enough to warrant their inclusion in the structure diagram. A formal analysis of each
secondary- and tertiary-structure base pair will be presented elsewhere.

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Figure 1C Escherichia coli 235 rRNA, 3' half.
Base pairs which contain strong comparative support are shown with a connecting symbol. Tick marks
designate A:U and G:C base pairs, a small closed dot is used for G:U pairs, a larger open circle denotes
A:G pairings, and a larger closed circle is used for all other noncanonical pairings. In contrast, conserved
nucleotides in the immediate vicinity of a convincing helix that can fonn canonical and G:U base pairs
are shown juxtaposed with no connecting symbol. These latter base pairings are (currently) neither proven
nor disproven by the comparative data.
One of the simple beauties of comparative sequence analysis, as it is practiced here, is that secondary-
and tertiary-structure base pairings are inferred in the absence of any knowledge of the principles of RNA

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structure. The best correlation is determined for every E.coli position in the alignment. This correlation
is based solely on the mutual change of nucleotides in this alignment, no matter what the underlying bases
are (e.g., this procedure will identify interchanges such as C:C H U:U as well as A:U H O:C). Thus,
canonical base pairs are not specifically searched for. The two positions that do correlate will do so
independently (to a first approximation) of flanking positions. Therefore the correlating pair could be
located in a multitude of structural environments. They could be antiparallei and adjacent to another set
of correlating positions (e.g., to fonn ahelix). arranged to form a set of paraliel interactions, isolated from
other known base pairings, etc. Secondary-structure helices can be nested or arranged into a pseudoknot
configuration. In the former, helices afe contained within one another, with a few long-range helices
establishing the primary structural domains. In the latter, the pseudoknol helix is formed by the crossing
of another helix.
As Nature will have it (as she usually does), the majority of all correlating pairs involve canonical and
G:U base pairs, the vast majority of all pairings are arranged into secondary-structure helices, and the
majority of these helices are nested. These expected observations should not be taken too lightly;
comparative sequence analysis has revealed the most basic principles of RNA structure.
In contrast with these predominant and expected reSUlts, we also observe some exceptions to these
common forms of RNA structure in the two rRNAs. Several unusual base pairings and base-pairing
interchanges are emerging from the comparative studies, including O:U H A:C, A:O H O:A, U:U H
C:C, and A:A H G:G noncanonical base pair replacements. While the majority of all base pairings are
adjacent and the nucleotides within them antiparallel, afew are isolated by themselves, and others contain
parallel base pairings. In addition, a small but growing number of helices cross the boundaries of others
helices, forming structural pseudoknots. These less common and structurally interesting elements both
contribute to our understanding of 16S and 23S rRNA structure and enrich our knowledge about possible
RNA structures. Are these exceptional and aberrant structural features biologically meaningful? Given
that our comparative studies have independently derived the basic rules for RNA structure, we are
confident that these newly proposed structural elements will be experimentally verified. A few pertinent
rRNA examples for these structural themes will be presented below.
B. CHARACTERISTICS OF THE BASE PAIRS
1, Canonical Pairings
Since all of the 16S and 23S rRNA secondary and tertiary structure base pairs are inferred from mutual
patterns of variation, and not from a search for canonical pairings, it is of interest to note the frequency
ofbase-pairtypes in these comparative structure models. Of the 16 possible pairing types (e.g., U:U, U:A,
U:O, U:C, C:U, C:A, etc.), the majority of all helical base pairs in the comparatively derived 16S and 23S
rRNA structures are composed of O:C and A:U pairings. In an analysis of hundreds of (eu)Bacterial and
chloroplast 16S and 23S rRNA sequences, the order of frequency for all of the comparatively inferred
base pairs are (with the relative frequencies for 16S and 23S separated by a slash): O:C [30%/28%J > C:O
[24%/28%J > U:A [15%1I3%J >A:U [12%/13%J > O:U [7%I7%J > u:o [6%/6%] > A:O [2%11 %] > O:A
[I%II%J > U:U [1 %/1%] > others (Konings and Outell, unpublished results). This result parallels the
thermodynamic stabilities for base pairs (Freier et a/. 1986; Turner, Sugimoto, and Freier 1988),
suggesting that the comparatively derived structures are, at least in part, selected to be thermOdynamically
stable.
Underlying these general base pair frequencies and constraints are different selection pressures for
each base pair position. While a detailed analysis is beyond the scope of this chapter, a few key
observations will be presented here. The majority of all base pair positions covary between canonical and
G:U pairings only. The others reflect varying amounts ofnoncanonical pairings. While canonical and G:U
pairings are still the predominant pairing type at these base pair positions, there are some positions that
only contain noncanonical pairs (Outell, Larsen, and Woese 1994; Konings and Outell, unpublished
results).
Next we ask ifthe secondary and tertiary structure base pairs are restricted in their variation. Ifahelical
base pair freely interchanges between all canonical and G:U pairings, then we can infer that this pairing
forms the standard base pairing conformation (assuming that the conformation of that base pair is
conserved across all sequences in the dataset). Its purpose is to maintain a helix and it is probably not
intimately involved in protein synthesis. However, if a base pair position is limited in the types of base
pairs observed, then we can surmise that this position might be part of a structural element that is

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important for recognition andlor function. [Because our rRNA database is now sufficiently large. we can
assume that each position has had ample opportunity to explore all possible nucleotides and base pairings.
Thus, if a position or a base pair is restricted in variation it is due to a structural or functional constraint
and not due to a small sampling size].
While many base pair positions appear to freely exchange between base pair types (e.g., A:U .... U:A
H C:G H G:C) without much restraint, and are thus under less selection pressure, a large number of
pairings are restricted in a variety of interesting ways. Within the (eu)Bacteria, some pairings are
invariant, although they differ in the other phylogenetic domains. Other base pair positions only inter-
change between two base pair types (e.g., A:U and G:C); while others exchange between three or more
base pair types. Base pairings that are predominantly R:Y-constrained (i.e., covary between A:U and G:C
or U:A and CoG) are relatively frequent, occurring at approximately 40% and 50% of the (eu)Bacterial
16S and 23S rRNA base pair positions, respectively. There is a tendency for these R:Y-restricted base
pairs to occur in long-range helices (e.g., helices that are separated by a large number of nucleotides) or
helices that have been associated with ribosomal function or ribosomal protein binding (Konings and
Gutell, unpublished). Of those base pairs with only two canonical pairing types, the majority involve
conservative transitional interchanges (Le., G:C H A:U). as we might expect. A small number of
canonical interchanges involve transversions. Of these, A:U H C:G and G:C H U:A occur less
frequently than G:C .... G:C and A:U .... U:A (Konings and Gutell, unpublished data). While the
conformational and functional significance of these latter canonical base pair exchanges are not currently
appreciated. we can speculate that they will be associated with protein binding, interesting RNA
conformations (e.g., base triples), and possibly ribosomal RNA function (e.g., tRNA binding). A more
detailed analysis of these base pair exchanges will be presented elsewhere (Konings and Gutell, manu-
script in preparation).
2, Noncanonical Pairings
In addition to the restricted canonical base pair exchanges, there afe several noncanonical helical base
pairings with interesting interchange patterns, The majority of these occur in secondary-structure helices.
although a few discussed in this section occur in more complex structural elements and might be
considered tertiary. For this discussion G:U base pairs will be considered to be noncanonical.
a. G:U Base Pairs and G:U H A:C Interchanges
The canonical base pairs G:C, CoG, U:A, and A:U are the most frequent comparatively derived pairings
(see above). Immediately following in frequency are G:U and U:G base pairs, occurring at 7% and 6%
in 16S and 23S rRNAs respectively [in a (eu)Bacterial and chloroplast alignment](Konings and Gutell,
unpublished results). Although this overall frequency is low, G:U base pairs occur at more than 30% of
the base pair positions in 16S and 23S rRNAs. The frequency of G:U pairings at these positions varies
over a large range. some positions containing just a few percent, while other positions are occupied with
nearly 100% G:U base pairings. Positions with a majority (> 50%) of G:U pairings have been classified
into three types (see Figure 3 in Gutell. Larsen, and Woese 1994). Those that are invariant (or nearly so)
within the (eu)Bacterial datasets are designated "I", Base pair positions that are predominantly G:U are
caBed "D" (for dominant). Within this class are examples of G:U .... U:G interchanges. The third type
has been called "N" for nontypical. Here the base pair interconverts between G:U and A:C. For the
positions where this occurs, it is usually quite pronounced within one or two phylogenetic groups, or
sometimes within parts ofa phylogenetic domain (e.g., within the gram positive group ofthe (eu)Bacteria).
At this time we will only discuss some examples of the "N" G:U type.
Although very infrequent, there are a few examples where the U:G (or CoAl is invariant in the three
primary phylogenetic domains, while U:G and C:A pairings covary in the mitochondria. The 16S rRNA
position 1402: 1500 contains a C:A juxtaposition in all (eu)Bacteria, Archaea, Eucarya, and the majority
of mitochondria, while a few phylogeneticaBy unrelated distantly related mitochondria have a U:G pair
(Gutell 1993b; Gutell, Larsen, and Woese 1994). The 16S rRNA position 1052:1206 is predominantly
U:G, with a few C:A pairings in the mitochondria (Konings and Gutell, unpublished). Although there is
no immediate explanation. it is of interest to note that both of these 16S rRNA base pairs are in immediate
proximity to sites associated with protein synthesis (Rinke-Appel et al. 1994; Prince et al. 1982;
Cunningham et al. 1993; Moine and Dahlberg 1994; Noller et al. 1990). Within 23S rRNA, positions
2249:2255 and 2457:2494 provide two more examples for this type ofU:G .... C:A interchange. The U:G

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pair is mostly invariant in the three phylogenetic domains for both of these base pairs. While U:G is the
predominant mitochondrial pairing at the 2249:2255 pcsition, there are a small but significant number of
sequences with a C:A pair. The second 23S base pair 2457:2494 contains approximately 50% C:A and
50% U:G base pairs in the mitochondria. Both of these positions are also in close proximity to sites
involved in protein synthetic activity (Noller et al. 1990; Sirum·Connolly and Mason 1993; Gregory,
Lieberman, and Dahlberg 1994). .
Some of the U:G H C:A interconversions occur primarily within one phylogenetic group, while
canonical pairings are present in the other phylogenetic groups. The 16S rRNA pair 249:275 interchanges
between U:G and C:A in the Archaea and (eu)Bacterial groups (Gutell, Larsen, and Woese 1994), while this
U:G pair alternates with otherpairs in the Eucarya and mitochondria. Two other examples ofthis mini-theme
occur at 16S rRNA pcsitions 1074:1083 and 1086:1099. At the 1074:1083 base pair, G:U and A:C pairs
covary in the Archaea, with the A:C pair present in over 80% of the Archaea sequences. Within the
(eu)Bacteria, this base pair is always a G:U, while the Eucarya and mitochondria maintain canonical pairings
along with the G:U pair. At the 1086: 1099 base pair, the (eu)Bacteria interchange between U:G and C:A
pairings, with the U:G pairoccuring in over 90% ofthe sequences. Canonical and other types ofnoncanonical
pairings are present in the Archaea, Eucarya, and mitochondria at the 1086: 1099 base pair. One last example
ofthis theme occurs in (eu)Bacteria at pcsitions 383:391 in 23S rRNA. This pairing is predominantly a C:A,
alternating to U:G in a few phylogenetically distinct organisms. This base pair and surrounding nucleotides
are deleted in the Archaea and Eucarya. These are the purest examples for this novel pairing exchange in
the rRNAs. Other alternating U:G H C:A pairs are not exclusive. Instead they also exchange with canonical
or noncanonical pairs. One prominent example is present at the (eu)Bacterial 16S rRNA base pair 152: 169.
Here the A:C pair exists in 75% of the sequences, followed by G:U(13%), G:G(6%), and N:N(6%). The
number of mutual changes (Le., covariations) occurring during evolution at this base pair position is quite
large. The most common compensatory change is between A:C and G:U base pairs, followed by A:C and
G:G base pairs. Other examples of alternating G:U and A:C base pairs within select phylogenetic groups
have been identified and will be presented elsewhere.
b. Purine:Purine, Pyrimidine:Pyrimidine, and Other Interconversions
Comparative studies have revealed several examples of A:G, G:A, A:A, G:G, U:U, and C:C pairings. A
fraction of these rare base pairs exchange with canonical pairings. However, each one of the noncanonical
base pairings is also involved in unique and exclusive covariations with other noncanonical pairings.
Although none of these has been explicitly associated with protein recognition. ribosomal function. or
more complex RNA-RNA interactions, we can speculate that each of them is involved in more than
simple base pairing.
A:G H G:A. There are several examples in the rRNAs for A:G H G:A covariations. In 16S rRNA
the cleanest example involves pcsitions 1357 and 1365, which are the closing base pairs for a hairpin loop
(Woese et al. 1983). Within the (eu)Bacteria, chloroplasts, and mitochondria, A:G and G:A interconvert
exclusively with one another, while in the Archaea and Eucarya this base pair consists only of canonical
base pairs. Three examples are known in 23S rRNA. The first occurs in a secondary-structure helix at
pcsitions 1858:1884 (Gutell 1993b; Trust et a1. 1994). This exchange is flanked on both sides with
noncanonical and canonical pairings. fonning an irregular helix. This unusual structural element only
forms in the (eu)Bacteria and chloroplasts (Gutell 1992). The Archaea and Eucarya have truncated this
part of the helix. The second 23S rRNA example, at pcsitions 2112:2169, does not occur in a secondary·
structure helix; instead. it is situated adjacent to other pairings that form a parallel structure (GuteH and
Woese 1990; see below). This structurally unique region is associated with the E site (Moazed and Noller
1989). Crosslinking studies have confirmed the propcsed 2112:2169 interaction (Doring, Gruer, and
Brimacombe 1991). The third 23S rRNA A:G H G:A exchange occurs in (eu)Bacteria at positions
857:920 (Konings and Gutell, unpublished). Although this pairing is flanked by a canonical pairing on
its 5' side and a G:U pairing on its 3' side, this helical region is irregular. with several A:G pairings. The
Archaea contain canonical pairings at 857:920 while the Eucarya have both canonical and noncanonical
pairs. The (eu)Bacterial 5S rRNA has an example of a A:G H G:A covariation between positions 75
and 101, which is nested in an irregular helical structure (Gutell, unpublished analysis). This base pair
is flanked on its 5' side with a U:G pair that exchanges with C:A and A:A pairings, and on its 3' side
with a G:G pair that interchanges with A:A (see below). Each of these A:G H G:A covariations is
found in an interesting structural context. One occurs at the end of a helix, one is present in a parallel

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structure. another is in close proximity to other A:G pairings. and two are flanked by other noncanonical
pairings.
U:U H CoCo There are three rRNA base pairings that covary exclusively between U:U and C:c. Two
of them occur in 16S rRNA. at positions 245:283 and 1307:1330. and one in 23S rRNA. at positions
1782:2586 (Outell and Woese 1990; Outell. Larsen. and Woese 1994). The 16S rRNA 245:283 base pair
is considered a lone pair since neither of the paired nucleotides is contiguous with an adjacent base pair.
This pair is a C:C in nearly all Eucarya sequences, while in both the (eu)Bacteria and Archaea there are
many documented U:U H C:C covariations. Note that this pairing is a few base pairs from the 249:275
U:O H C:A exchange (see above). Ribosomal protein SI7 protects these base pairs from modification
(powers and Noller 1995). suggesting that these unusual pairings are important for protein recognition.
The second 16S rRNA U:U H C:C exchange occurs at the 1307: 1330 base pair (Outell and Woese 1990;
Outell. Larsen. and Woese 1994). This covariation is present within each of the three phylogenetic
domains. and is flanked on its 3' side by the 1308·1314/1323·1329 helix and on its 5' side with several
highly conserved O:A and A:O juxtapositions. The third U:U H C:C exchange associates the 23S rRNA
positions 1782 and 2586. Not only is this pairing isolated from other known base pairings (e.g.• a lone
pair; see below), it is also a long-range interaction between domains N and V.This long range covariance
is supported by crosslinking studies (Stiege. Glotz. and Brimacombe 1983; Mitchell et al. 1990).
A:A H G:G. The 16S rRNA lone base pair at positions 722:733 covaries between A:A and 0:0
(Outell and Woese 1990). This base pair is considered a lone pair since both of the paired nucleotides
are not contiguous with an adjacent base pair. A second example of this type of exchange occurs in the
(eu)BacteriaI5S rRNA at positions 76: 100. as noted above. This pair is adjacent to a A:O H O:A pairing
exchange on it's 5' side; an invariant U:A pair is on its 3' side. Here both examples of the A:A H G:G
covariance occur in an irregular helical structure, suggesting a recognition motif (Gutell, Larsen, and
Woese 1994).
Other interconversions. There are also a few base pair positions that alternate between a noncanonical
and canonical base pair.Those that interchange primarily between two types of base pairings are noted here.
U:A H G:G at 16S rRNA positions 438:496 (Outell and Woese 1990). This pairing constraint occurs
in (eu)Bacteria and Archaea. The Eucarya do not have an analogous helix. This pairing is situated within
a putative base triple (see below; Outell. Larsen. and Woese 1994).
A:C H U:A at 16S rRNA positions 996:1045 (Woese personal communication; Outell. Larsen. and
Woese 1994). This base pair is only present in the (eu)Bacteria. The Archaea and Eucarya have a different
helical fonn in the corresponding region. This pairing is at the end of a helix and involved in a putative
base triple (Outell et al.. mss. in preparation).
A:G H R:U at 16S rRNA positions 122:239 (Outell and Woese unpublished). This exchange only
occurs in the (eu)Bacteria. The predominant alternation is between the O:U and A:O base pairings. A:U
pairings are the third most frequent base pair. interchanging mostly with the O:U pairing. The Archaea
contain mostly A:O base pairs. while the Eucaryal helix and base pair are not strictly homologous. The
122:239 base pair is at the terminus of the 122·1291232·239 helix. and situated in close proximity to a
putative triple between positions 121 and 1241237 or 1251236 (see below; Outell et al.. mss. in preparation).
A:G H G:U at 23S rRNA positions 15:525 and 2675:2732. The 15:525 O:U H A:O base pair
covariance is strongest in the (eu)Bacteria, present but weaker in the Archaea, and nonexistent in the
Eucaryal domain. The 2675:2732 base pair alternates between A:O and O:U in the (eu)Bacteria. The
Archaea and Eucarya are lacking this base pair; instead. they have an extra base pair at the terminus of
the 2646·265212668·2674 helix. This 2675:2732 interaction is at the junction of a putative coaxial stack
between helices 2646·265212668·2674 and 2675·268012727·2732 (see below; Oute1l1992; OutellI993b).
The significance of these constrained and noncanonical base pair alternations is not currently under-
stood. However, it is of interest to note that the majority of them occur in a unique structural context, be
it at the end of a helix. in a lone pair, in proximity or direct association with (putative) base triples, or
at the junction of a (putative) coaxial helix stack. We can speculate that these unusual pairings and pairing
exchanges are generally important for the fonnation of specialized structural confonnations. We await
the experiments that should enlighten us as to why these base pairings are restricted in their variation.
C. ARRANGEMENT OF THE BASE PAIRS AND CHARACTERISTICS OF HELICES
1. Traditional Organization of Base Pairs and Helices
Comparative sequence analysis, in its simplest fonn, identifies positional covariation. From these
correlations, we infer base pairings. As noted earlier, the basic principles of base pairings in RNA have

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been derived independently in these comparative studies. It was also noted that the majority of the
canonical base pairings are arranged consecutively and antiparallei with one another so as to form helices.
Further, the majority of these helices are constructed so that each helix is within the boundaries of another
helix (e.g.. they are not knotted), In contrast. to traditional base pairs and helices, a few base pairs are not
flanked by other base pairings (e.g., lone pairs), a small number of helices cross the boundaries of other
helices fanning pseudoknots, while other base pairs are arranged in parallel. The nontraditional structural
elements are described, along with a few other interesting structural features derived from our campara·
tive studies of rRNA, in the following paragraphs.
2. Lone Pairs
Approximately 20 comparatively derived base pairings are not components of a secondary or tertiary
structure helix. Rather, these base pairs are isolated from other base pairings (see Figure I). While a few
of the lone pairs are completely isolated from other base pairings (i.e., they are not within a few nucleotides
of the closest base pair; see below), most of the lone pairs are adjacent on their 5' or 3' sides to an existing
secondary structure helix. While some of these lone pairs contain noncanonical base pairings (see above).
the majority were identified by canonical base pairing exchanges. Examples of lone pairs that are completely
isolated from other base pairs are in the 16S rRNA, 450:483 and 1399:1504, and in the 23S rRNA,
1782:2586 (noncanonical pair, see above) and 2117:2172 (see below). Lone pairs that are partially isolated
in 16S rRNA are: 47:361; 245:283 (noncanonical pair, see above); 575:880; 722:733 (noncanonical pair,
see above); and 779:803. Partially isolated lone pairs in the 23S RNA are: 30:510; 61:93 (see below); 67:74;
234:430; 319:323; 1082: 086; 087: I 02; 1262:2017; 1752:1756; 1800: 1817; 2282:2427; 2512:2574; and
2626:2777 [Lone pairings that cross an existing helix (Le., a pseudoknot) are designated in bold type). Three
of the 23S rRNA lone pairs form a hairpin loop composed of three nucleotides (319:323; 1082:1086; and
1752: 1756). Each of these is immediately contiguous on its 5' and/or 3' sides with other helical base pairs.
All of these single base pairs, in isolation from other interactions, are probably not very stable. Thus for these
base pairs to form, we suspect that auxiliary factors, such as proteins and/or other RNA:R.!'1A interactions
(i.e., stacking onto adjacent bases or base triple formation) stabilize their formation. The 2282:2427
interaction has been substantiated by crosslinking experiments (Mitchell et al. 1990).
3. Pseudoknots
As noted earlier, the majority of all base pairs and helices are nested; their formation does not involve
the formation of a knot. In contrast, pseudoknots are helical interactions that cross the boundaries of
another set of helices. There are approximately 15 of these structural motifs in the 16S and 23S rRNAs
(Gutell, Larsen, and Woese 1994 and references therein). The three pseudoknot helices in 16S rRNA are:
17·19/916·918; 505-507/524-526; and 570-571 /865-866 (see Figure I). The base pairings in these helices
are all canonical. These helices appear to be more than just a structural entity. While the structural
integrity of each of these comparatively derived helices has been experimentally substantiated, they have
also been shown to be important for protein synthesis (Brink, Verbeet, and de Boer 1993; Powers and
Noller 1991; Vila et al. 1994). These pseudoknots are all located in close proxintity to the helices that
establish the three structural domains in 16S rRNA and in regions ofthe rRNA that are conserved in the
Archaea. Eucarya. and (eu)Bacteria. We can speculate that they might coordinate interactions, and even
movement. between the 16S rRNA structural domains.
The 23S rRNA contains more than ten pseudoknots. The majority of the base pairs in these structural
elements are canonical, although there are a few noncanonical pairings. While the pseudoknot helices in
16S rRNA are two or three base pairs in length, some of the 23S rRNA pseudoknots consist of only a
single base pair (e.g., 2626:2777). However, as with 16S rRNA, the longest pseudoknots are only three
base pairs in length. Two of the 23S rRNA pseudoknots associate two hairpin loops (designated below
as "loop-loop" interactions). The 23S rRNA pseudoknots are: 61:93 and 65-66/88·89 (loop-loop); 67:74;
234:430; 317-318/333-334; 413-416/2407-2410 (loop-loop); 1005-100611137-1138; 1343-134411403-
1404; 2111/(2144 2147); 2112:2169 (noncanonical, see above), 2113:2170, 2117:2172 (base pairings in
parallel, see below); 1782:2586 (noncanonical, see above); 2328-2330/2385-2387; and 2626:2777. A few
of the comparatively inferred interactions in 23S rRNA are also substantiated with experimental data.
Recently, the proposed 1005-100611137-1138 pseudoknot helix in domain II has been verified by site-
directed mutagenesis and shown to be important for ribosomal function (Rosendahl, Hansen, and
Douthwaite, 1995). Domain III of23S rRNA contains the 1343-134411403-1404 pseudoknot helix, which
is positioned at the base of three secondary structure helices. Experiments verified this pairing and revealed

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that this helix is important for ribosomal protein binding (Kooi et al. 1993). Experimental support for the
1782:2586 and 2112:2169 interactions was discussed earlier. Direct experimental evidence (eg. crosslinking)
for the other proposed 235 rRNA pseudoknots is currently lacking. However, it should be emphasized that
the comparative support (i.e., the number of compensatory base changes) for each of these pseudoknot
interactions is very strong. Thus, we anticipate experimental verification in time.
In summary, all of the proposed rRNA pseudoknot helices are short, ranging from one to three
basepairs in length. The majority of these helices have either their 5' or 3' halves immediately adjacent
to the end of a secondary-structure helix. And some of the pseudoknot helices can potentially stack onto
more than one secondary-structure helix. These observations, in parallel with the experimental charac-
terization of a simple pseudoknot structure that revealed coaxial stacking of the stems (Puglisi, Wyatt,
and Tinoco, 1990), lend support to the idea that some of the 165 and 235 rRNA pseudoknot helices can
be coaxially stacked onto one or more adjacent helices in a confonnationally static or dynamic manner
(ten Dam, Pleij, and Draper 1992, see below).
4, Base Pairings in Parallel
The majority of all base pairs are positioned antiparallel to one another to form traditional RNA helices.
In contrast, there are a few adjacent base pairs that are configured in parallel to one another. These occur
in 235 rRNA at positions 2112/2169, 2113/2170, and 211712172 (Gutell and Woese 1990; Gutell, Larsen,
and Woese 1994). These pairings are at the E site (Moazed and Noller 1989), suggesting that this unusual
arrangement of base pairs is associated with protein synthesis. As noted earlier, positions 2112 and 2169
alternate primarily between A:G and G:A pairings, and the proposed interactions have been validated by
cross-linking data (Doring, Greuer, and Brimacombe 1991).
5. Base Triples
Our search for base triples by comparative methods has led to several unexpected findings. Initially, no
strong 165 or 235 rRNA triple candidates were identified. Consequently we investigated the triples in
tRNA for which there are several three-dimensional crystal structures available for different tRNAs.
Moreover. the comparative sequence alignment database is large and representative of all tRNA classes.
First, these studies revealed that base triples are not conserved across all tRNAs. For example. in yeast
lRNA"", the three triples involve positions 45(10:25), (12:23)9, and (13:22)46. In E.coli tRNAGI", the one
and only triple associates positions 45(13:22), while in E.co/i tRNA"" the base triples form between
positions 9(13:22), 8(14:21), and 48(15:20) (Gautheret, Damberger, and Gutell 1995, and references
therein). Second. similar base triple conformations can fonn in the absence of covariation. For example.
the conformation for the (12:23)9 lRNA base triple is nearly identical for the sequences (U:A)A and
(U:A)G (Klug, Ladner, and Robertus 1974; Gautheret, Damberger, and Gutell 1995). Third, the base pairs
in the helices that are associated with base-triple fonnation tend to have a strong neighbor effect. In
particular we observe that paired nucleotides have a strong correlation with nucleotides in the flanking
base pairs and reflect a strong correlation with its base pair partner as well. Taken together, these findings
suggest that for base triples, the structural unit under selection is not simply and only the base pair, as
it is for base:base interactions (to a first approximation). For base triples, a larger three-dimensional
structure appears to be evolving as a unit under the same selection constraint (Gautheret. Damberger, and
Gutell 1995). These findings help explain in part why our initial searches for base triples in 165 and 235
rRNA were inconclusive.
This analysis of the lRNA base triples has resulted in our development of quantitative methods to
better identify these structural features (Gautheret, Damberger, and Gutell 1995). The newer comparative
algorithms have improved our identification of the known base triples in lRNAs and group I introns, and
have also suggested new base triples in the group I introns (Gautheret, Damberger, and Gutell 1995).
More recently these methods have been applied to the 165 and 235 rRNA datasets, resulting in several
strong base triple candidates (Gutell et aI., manuscript in preparation). A few of them are mentioned here.
Previously we had recognized a 'probable triple covariance' involving positions 440, 494, and 497 in 165
rRNA (Gutell, Larsen, and Woese 1994). This triple is detected with the newer computational methods,
suggesting that it is indeed a significant correlation. However, this pairing is a bit unusual. Although
positions 440 and 494 are juxtaposed in the secondary structure. the correlation is weaker than for
positions 440 and 497. Within the boundaries of this putative base triple lies the unusual base pair 438:496
(see above), which alternates between U:A and G:G (Gutell and Woese 1990). Taken together, these two
sets of correlations suggest an interesting structure at the base of the 442-446/488-492 helix. Our

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quanititive algorithms have also identified a strong correlation between the unpaired 16S rRNA position
121 and the 124:237 and 125:236 basepairs, suggesting another probable base triple. While position 121
can potentially interact with either base pair, the base triple more likely involves just one of these base
pairs. The other correlating base pair is probably due to a very strong neighbcr effect (we observe a strong
neighbcr effect in the tRNA D-helix where three of the four base pairs are involved in base triples; Gutell
et al. 1992; Gautheret, Damberger, and Gutell 1995). Several other base triples have been detected that
also link an unpaired nucleotide with a base pair that is afew nucleotides away. The details for these base
triples will be presented elsewhere (Gutell et aI., manuscript in preparation).
Within 23S rRNA there are several excellent base triple covariations (Gutell et aI., manuscript in
preparation). A few of these incorporate two distant regions of the secondary structure that have
previously been physically linked by crosslinking experiments (Mitchell et al. 1990; Doring, Greuer, and
Brimacombe 1991). Probably the most tantalizing of these involves position 746 and the 2057:2611 base
pair. Here the triplet (G:C)U is most frequent, followed by (A:U)G and (C:G)e. Positions 748 and 2613-
2614, in very close proximity to these triple nucleotides. have been crosslinked with ultraviolet irradiation
(Mitchell et al. 1990), strongly suggesting the authenticity for this comparatively derived base triple. This
base triple, if real, might well be involved in protein synthesis. The 2057:2611 base pair adjoins the
peptidyJ transferase loop and is implicated in resistance to numerous antibiotics (reviewed in: Douthwaite
1992). In addition, chemical protection studies have mapped vemamycin B to position 752 (Moazed and
Noller 1987). Other putative 16S and 23S rRNA base triples have been identified. These along with the
base triples discussed here will be presented in greater detail in the near future (Gutell et aI., manuscript
in preparation).
6. Coaxial Helices
Ultimately. we seek to fold the secondary-structure helices into a three-dimensional structure. The few
known tertiary interactions and pseudoknot helices already impose a sense of three dimensionality upon
the 16S and 23S rRNA structures. However. many more constraints are necessary to achieve a biologi-
cally meaningful structure. Potentially, a better understanding of how adjacent helices interact could
provide some of this infonnation.
Our knowledge of the tRNA crystal structure (Kim 1979) and physical investigations of small
oligonucleotides (Puglisi, Wyatt, and Tinoco 1990) reveal that adjacent helices can coaxially stack,
forming an elongated helical structure. Inspection of the 16S and 23S rRNA secondary-structure diagrams
(Figure 1) suggests many possible helical stackings. Can we determine which of these are present in the
native 16S and 23S rRNA structures? In 1983, Carl Woese proposed a comparative rationale that could
potentially identify some helices that are coordinated into an elongated helical structure (Woese et a1.
1993). Since two helices that are coaxially stacked are expected to maintain a constant overall length,
comparative support for coaxial helices would be derived from cases in which one helix in a group of
organisms is shorter. while its coordinating helix in that group is longer by the same amount thereby
maintaining the same combined length. TIlls is noted in two sets of helices proposed to fonn coaxial
stacks, one in 16S and the otherin 23S rRNA. The 16S rRNA helices 500-504/541-545 and 511-515/536-
540 are together 10 base pairs in length. In the (eu)Bacteria the two helices are 5 and 5 bp in length. In
contrast, the corresponding helices in both the Archaea and Eucarya are 6 and 4 bp in length (Winker and
Woese 1991). A pseudoknot helix forms between the side bulge ofthese two helices and the large hairpin
loop capping the 511-515/536-540 helix (see above), suggesting a more complex and dynamic structure.
At the base of the a-sarcin helix in 23S rRNA, helix 2646-265212668-2674 is proposed to stack onto the
3' adjoining helix, 2675-268012727-2732, forming a 13-bp coordinated helix. In the (eu)Bacteria lengths
of the helices are 7 and 6 bp, while in the Archaea and Eucarya the corresponding lengths are 8 and 5
bp (see abcve discussion about A:G H G:U interconversions, Gutell 1992; Gutell 1993b).
7, Tetraloops
The search for positional covariation has identified base pairs, and has revealed much detail about pairing
constraints and the arrangement of the base pairs into larger structural elements. While the analysis of
unpaired nucleotides is not as advanced. these studies have categorized the unpaired nucleotides into
several categories: hairpin loops, bulges. terminal extensions of helices, internal loops, and multistem
loops. Only the most frequent of the hairpin loops, the so called "tetraloops" will be discussed here.
The most frequent hairpin loop size in 16S and 23S rRNA is four. These tetraloops account for
approximately 50% and 40% of all hairpin loops in prokaryotic 16S and 23S rRNAs, respectively. Of the

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256 possible sequences of length four. the majority of the rRNA tetraloops are distributed into three
sequence families - GNRA. UUCG. and CUUG (Woese et al. 1990). The tetraloop at positions 83-86 in the
165 rRNA alternates almost exclusively between the sequences CUUG. UUCG. and GeAA. The particular
closing base pair for these hairpin loops is usually associated with the tetraloop sequence. CUUG is
predominantly closed with a G:C pair. UUCG is closed with C:G. while the closing base pair for the GCAA
tetraloop is usually an A:U. While this correlation between the closing base pair and the type of tetraloop
is. on the whole. true for other UUCG and CUUG tetraIoops in the rRNA. the other GNRA tetraloops are
usually closed with a C:G or G:C pair (Woese et al. 1990). A few tetraloops alternate between these three
sequence families, but the majority of these positions are evolving more slowly and are usually restricted
to a smaller number of sequence motifs. The GNRA sequence family accounts for the majority of tetraloop
positions that are conserved at alevel above 80%. The various tetraloop themes suggest that these structural
elements can serve different functions in the rRNA structure.
Do tetraloops influence the folding of rRNA. that is. does their stability contribute to nucleating the
formation of important helices? Do they form a unique conformation and/or interact with other regions
of the RNA? The UUCG loop. closed with a C:G base pair is very stable (Tuerk et al. 1988). which helps
to explain why it is a frequent sequence motif in the rRNAs. However. the GNRA sequence motif occurs
more frequently than UUCG even though this tetraloop is less stable (although they are slightly more
stable than sequences that are less frequently observed in rRNA tetraloops) (Antao. Lai. and Tinoco 1991;
5antaLucia. Kierzek and Turner 1992). Why are the GNRA tetraloops so abundant if they are not as
energetically stable? While the conformations for both of these tetraloops are very compact and structur-
ally unique (Varani. Cheong. and Tinoco 1991; Heus and Pardi 1991). these features do not directly
resolve the question. Thus, are the GNRA tetraloops selected to interact with other regions of the rRNA
or to be recognized by proteins? One study has revealed the formation of a base triple between the third
nucleotide of the GNRA loop and a helical base pair (Jaeger. Michel. and Westhof 1994). In another
study. an internal loop comprised of an 11 nucleotide motif was shown to bind to GAAA hairpin loops
(Costa and Michel 1995). In other cases. it has been shown that certain proteins recognize specific GNRA
tetraloops (Orita et aL 1993; 5zewczak et al. 1993). Moreover. !X-sarcin recognition of the GAGA
tetraloop requires a C:G closing base pair (Gluck. Endo. and Wool 1994). suggesting a rationale for some
of the closing base pair constraints noted earlier. We can only speculate that some tetraloops nucleate
folding so as to assure the proper rRNA structure (e.g.• the 165 rRNA 83-86 tetraloop). while others.
including some of the conserved GNRA motifs, are involved in tertiary interactions. Consistent with this
proposition, there are two possible tertiary interactions in 16S rRNA that involve a tetraloop. The first
is between position 1268. the third nucleotide of a GNRA loop and the base pair 1311: 1326 (Gutell.
unpublished). This putative base triple is similar to the form first recognized by Michel in group I introns
(Jaeger. Michel. and Westhof 1994). When the R (of the GNRA loop) is a G. then the interacting base
pair is an A:U, and when it is an A, the interacting base pair is a G:C. A second tertiary interaction
involving a 165 rRNA tetraloop has been known since 1985 (Gutell et al. 1985. Gutell. Noller. and Woese
1986). The unpaired position 570 covaries with 866. the last nucleotide of the 863-866 tetraloop. While
E.coli has a UAAC loop sequence. many 165 rRNA sequences contain a GAM sequence in the
corresponding hairpin loop (Gutell 1993b). Do GNRA telraloops have a special conformation that
predisposes them to tertiary interactions? The answer is yes. Very recently it has been determined that
GNRA tetra100ps can adopt the uridine turn conformation (Jucker and Pardi 1995). This U-turn. as it is
known. creates a sharp turn in the backbone. preparing the nucleotides immediately 3' for tertiary-like
basepairing (Jucker and Pardi 1995). We can now begin to understand how positions 865 and 866 in the
UAAC and GAAA tetraloops can form a pseudoknot with positions 570-571. These recent findings and
the high frequency of GNRA tetraloops in 165 and 235 rRNAs now suggest that other GNRA and related
tetraloops may well be involved in tertiary interactions.
Ill. A COMPARATIVE PERSPECTIVE ON THE STRUCTURE OF rRNA
Just a few years ago. we began our studies ofrRNA structure with a small collection of aligned rRNA
sequences, a minimal knowledge of how RNA folds up into specific secondary and tertiary structures,
and little appreciation for the functionally important structural elements. In addition, we accepted the
simple principle that different sequences can adopt a similar secondary and tertiary structure when all of
the members of the sequence family under study are constrained to a common three-dimensional
structure. With this sequence information and conceptual framework, comparative sequence analysis has

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transfonned the 16S and 23S rRNA datasets into reliable secondary-structure models and the beginnings
oftertiary-structure models. At present our inquires are leading to the recognition ofnovel RNA structural
elements while providing an important perspective on how the rRNAs are involved in the functioning
ribosome.
The comparatively derived 16S and 23S rRNA secondary- and tertiary-structure models are the result
of over 10 years of development. Iri the early 1980" with a handful of 16S and 23S rRNA sequences,
the search for a common secondary structure relied primarily on the identification of covarying nucle-
olides present in a secondary-structure helix. These efforts produced the initial secondary-structure
models for 16S and 23S rRNA. While, at the time, our confidence in these models was based on a minimal
amount of comparative evidence. the majority of the secondary~structure base pairings originally pro-
posed are present in today's highly refined secondary-structure diagrams. However, the older compara-
tive methods and the limited number of 16S and 23S rRNA sequences available were only sufficient to
establish the basic secondary-structure models. No tertiary interactions could be discerned. nor could we
begin to understand other structural and confonnational details.
At this time we have a very large collection of 16S and 23S rRNA sequences and a better appreciation
for the comparative sequence paradigm, along with more powerful and generalized algorithms for
correlation analysis. With the assistance of a faster computer, we are now detennining the best correlation
for each position in the 16S and 23S rRNA. This exhaustive study has already yielded a highly refined
secondary-structure model and led to the identification of numerous tertiary interactions which have an
overwhelming degree of comparative support.
For the future we wonder whether the comparative sequence paradigm will have yet more to offer in
regard to understanding rRNA structure. The answer is surely affinnative. We should expect comparative
analysis to reveal more tertiary interactions, including base triples, and to provide a better understanding of
the relationships between sequence and structure (e.g., tetraloops) with the goal of ascertaining new RNA
structure motifs. Moreover, comparative analysis should pennit us to further investigate patterns ofvariation
and how they relate to RNA confonnation (e.g., a O:A pairing motif in internal loops, Oauthere~ Konings,
and Outell 1994). Ultimately, comparative analysis and the effort to determine a structure common to all
of the 16S and 23S rRNA sequences will show us more than one-to-one positional covariance. These
methods will take advantage of the growing appreciation for RNA confonnations and the mapping between
a sequence and its secondary and tertiary interactions to assist in the search for a common structure.
ACKNOWLEDGMENTS
I would like to gratefully acknowledge the contribution and influence Drs. Carl Woese and Harry
Noller had on this chapter. Critical readings and enhancements by Drs. Al Dahlberg and Bob Zimmennann
are appreciated.
REFERENCES
Antao V.P., Lai S.Y. and Tinoco I. Jr. (1991). A thennodynamic study of unusually stable RNA and DNA hairpins. Nucleic
Acids Res. 19:5901-5905.
Branlant C., Krot A., Machatt M.A., Pouyet J., Ebel J.P., Edwards K., and Kossel H. (1981). Primary and secondary
structures of Escherichia coli MRE 600 23S ribosomal RNA. Comparison with models of secondary structure for
maize chloroplast 23S rRNA and for large portions of mouse and human 16S mitochondrial rRNAs. Nucleic Acids
Res. 9:4303-4324.
Brink M.F., Verbeet M.Ph., and de Boer H.A. (1993). Ponnation of the central pseudoknot in 16S rRNA is essential for
initiation of translation. EMBO 1. 12:3987-3996.
Brosius J ., Palmer M.L., Kennedy, PJ., and Noller H.F. (1978). Complete nucleotide sequence ofa 16S ribosomal RNA
gene from Escherichia coli. Proc. Natl. Acad. Sci. USA 75:4801-4805.
Brosius J.. Dull T., and Noller H.F. (1980). Complete nucleotide sequence of a 23S ribosomal RNA gene from Escherichia
coli. Proc. Nad. Acad. Sci. USA 77:201-204.
Brosius J .. Dull T•• Sleeter D.D., and Noller H.F. (1981). Gene Organization and Primary Structure of a Ribosomal RNA
Operon from Escherichia coli. 1. Mol. Bioi. 148:107-127.
Costa M. and Michel F. (1995). Frequent use of the same tertiary motif by self·fo1ding RNAs. EMBO 1. 14:1276-1285.
Doring T., Gruer B., and Brimacombe R. (1991). The three-dimensional folding of ribosomal RNA; localization of a series
of intra-RNA cross-links in 23S RNA induced by treatment of Escherichia coli 50S ribosomal subunits with bis-(2-
chloroethyl)-methylamine. Nucleic Acids Res. 19:3517-3524.

16. -
•
126
Freier S.M., Kierzek R.t Jaeger J.A., Sugimoto N., Caruthers M.H., Neilson T., and Turner D.H. (1986). Improved free-
energy parameters for predictions of RNA duplex stability. Proc. Nat/. Acad. Sci. USA 83:9373-9377.
Gautheret D., Konings D., and GuteH R.R. (1994) A major family afmotifs involving G.A mismatches in ribosomal RNA.
J. Mol. Bioi. 242:1-8.
Gautheret D., Damberger S.H., and Gulell R.R. (1995) Identification of Base-triples in RNA using Comparative Sequence
Analysis. J. Mol. BioI. 248:27-43.
Glotz C., Zwieb C., and Brlmacombe R. (1981). Secondary structure of the large subunit ribosomal RNA from Escherichia
coli. Zea mays chloroplast. and human and mouse mitochondrial ribosomes. Nucleic Acids Res. 9:3287-3306.
Gluck A., Endo Y., and Wooll.G. (1994), The ribosomal RNA identity elements for ricin and for alpha-sarcin: mutations
in the putative CG pair that closes a GAGA tetraloop. Nucleic Acids Res. 22:321-324.
Gregory S.T., Lieberman K.R., and DahlbergA.E. 1994. Mutations in the peptidyl transferase region ofE.co/i 23S rRNA
affecting translational accuracy. Nucleic Acids Res. 22:279-284.
Gutell R.R., Welser B., Woese C.R., and Noller H.F. (1985). Comparative anatomy of 16S-like ribosomal RNA. Progress
in Nucleic Acid Research and Molecular Biology 32:155-216.
Gutell R.R.. Noller H.F., Woese C.R. (1986) Higher order structure in ribosomal RNA. EMBO J. 5: 1111-1113.
Gulell R.R. and Woese C.R. (1990) Higher order structural elements in ribosomal RNAs: Pseudo-knots and the use of
noncanonical pairs. Proc. Natl. Acad. Sci. USA 87:663-667.
GuleU, R.R., Power, A.. Hertz, G.Z., Putz, EJ. and Slonno, G.D. (1992) Identifying Constraints on the Higher·Order
Structure of RNA: Continued Development and Application of Comparative Sequence Analysis Methods. Nucleic
Acids Res. 20:5785-5795.
Gutell R.R. (1992) Evolutionary Characteristics of 16S and 23S rRNA Structures.pp. 243-309. PRESENTED AT: The
Symposium - The Origin and Evolution of Prokaryotic and Eukaryotic Cells. Shimada. Japan. April 22-25. 1992.
Hyman Hartman and Koichiro Matsuno (eds.). World Scientific Publishing Co.• New Jersey. USA
Guten R.R.. Gray M.W., and Schnare M.N. ( 1993) A compilation of large subunit (23S- and 23S·like) ribosomal RNA
structures. Nucleic Acids Res. 21:3055-3074 [Database issue].
Gulell R.R. (l993a). Comparative studies of RNA: inferring higher·order structure from patterns of sequence variation.
Current Opinion in Structural Biology 3:313-322.
Gutell R.R. (l993b) The Simplicity Behind the Elucidation of Complex Structure in Ribosomal RNA.p.477-488. In The
TranslatioMI Apparatus. Nierhaus et aI.• eds. Plenum Publishing Corporation.
GUlell R.R. (1994) Collection of SmaJl Subunit (16S- and 16S·like) ribosomal RNA structures: 1994. Nucleic Acids Res.
22:3502-3507(Database issue).
Gutell R.R., Larsen N., and Woese C.R. (1994). Lessons from an evolving rRNA: 16S and 23S rRNA structures from a
comparative perspective. Microbiological Reviews 58:10-26.
Heus H.A. and Pardi A. (1991). Structural Features That Give Rise to the Unusual Stability of RNA Hairpins Containing
GNRA Loops. Science 253:191-194.
Hill W.E., Dahlberg A., Garrett R.A.. Moore P.B., Schlessinger D., and Warner J.R. eds. (1990). The Ribosome:
Structure. Function &. Evolution. Hill et al., eds. American Society for Microbiology.
Holley R.W., Apgar J., Everett G.A., Madlson J.T., Marqulsee M., Merrill S.H., Penswick J.R., and Zamir A. ( 1965).
Structure of a Ribonucleic Acid. Science 147:1462-1465.
Jaeger L, Michel F., and Westhof E. (1994). Involvement of a GNRA Tetraloop in Long·range RNA Tertiary Interactions.
J, Mol. BioI. 236:1271-1276.
Jucker F.M. and Pardi A. (1995). GNRA tetraloops make a U-turn. RNA 1:219-222.
Kim SoH. (1979). Crysta1 Structure of Yeast tRNA-phe and General Structural Features of Other tRNAs. pp.83-100 In
Transfer RNA: Structure. Properties. and Recognition. Schimmel P.R.. Soil D.• and Abelson l.N., eds, Cold Spring
Harbor Laboratory,
KJug A., Ladner J., Robertus J.D. (1974). The Structural Geometry of Co-ordinated Base Changes in Transfer RNA. J.
Mol. Bioi. 89:511-516.
Kooi, E.A., Rutgers C.A., Mulder A., Van't Riel J., Venema J., and Raue H.A. (1993). The phylogenetically conserved
doublet tertiary interaction in domain 1II of the large subunit rRNA is crucial for ribosomal protein binding. Proc. Nad.
Acad, Sci. USA 90:213-216.
Levitt M. (1969). Detailed Molecular Model for Transfer Ribonucleic Acid. Nature 224:759-763.
Madison J.T., Everett G.A., and Kung H.K. (1966). On the Nucletide Sequence of Yeast Tyrosine Transfer RNA. pp. 409-
416. Volume XXXI Cold Spring Harbor Symposia on Quantitative Biology
Maidak B.L., Larsen N., McCaughey M,J., Overbeek R., Olsen G,J., Fogel K., Blandy J., and Woese C.R. (1994). The
Ribosomal Database Project. Nucleic Acids Res. 22:3485-3487 [Database issue].
Mitchell P., Osswald M., Schueler D., and Brimacombe R. (1990). Selective isolation and detailed ana1ysis ofintta-RNA
cross-links induced in the large ribosomal subunit of E.coli: a model for the tertiary structure of the t.RNA binding
domain in 23S RNA. Nucleic Acids Res. 18:4325-4333.
Moazed D. and Noller H.F. (1987). Chloramphenicol. erythromycin, carbomycin and vernamycin B protect overlapping
sites in the peptidyl transferase region of 23S ribosomal RNA. Biochimie 69:879-884.

17. -
I
127
Moazed D. and Noller HS. (1989), Interaction oftRNA with 235 rRNA in the Ribosomal A, P. and E Sites. Cell 57:585-
597.
Nierhaus K.H., Franceschi F., Subramanian A.R., Erdmann V.A., Wittmann-Liebold B. (1993), The Translational
Apparatus. Nierhaus et aI., eds. Plenum Publishing Corporation.
Noller H.F., Kop J., Wheaton V., Brosius J., GuteH R.R., Kopylov A.M., Dohme F., Herr W.o Stahl D.A., Gupta R.,
and Woese c.R. (1981), Secondary structure model for 235 ribosomal RNA. Nucleic Acids Res. 9:6167--6189.
Noller H.F., Moazed D., Stem S., Powers T., Allen P.N., Robertson J.M., Welser B. and Triman K. (1990). Structure
of rRNA and lis Functional Interaction in Translation. pp. 73-92. In The Ribosome: Structure. Function & Evolution.
Hill et aI.• eds. American Society for Microbiology. Washington. DC.
Olsen GJ. (1983). Comparative Analysis of Nucleotide Sequence Dara. Ph.D. thesis. University of Colorado.
Orita M., Nishikawa F., Shimayama T., Taira K., Endo Y., and Nishikawa S. (1993). High-resolution NMR study of
a synthetic oligoribonucleotide with a tetranucleotide GAGA loop that is a substrate for the cytotoxic protein. ricin.
Nucleic Acids Res. 21 :5670-5678.
Powers T. and Noller H.F. (1991). A functional pseudoknot in 16S ribosomal RNA. EMBO 1. 10:2203--2214.
Powers T. and Noller H.F. (1995). Hydroxyl radical footprinting of ribosomal proteins on 16S rRNA. RNA 1: 194-209.
Prince J.B., Taylor B.H., Thurlow D.L., OCengand J., and Zimmennann R.A. (1982) Covalem crosslinking of tRNA-
val to 16S RNA at the ribosomal P site: identification of crosslinked residues. Proc. Nat/. Acad. Sci. USA 79:5450-
5454.
Puglisi J.D., Wyatt J.R., and Tinoco IJr. (1990). Conformation of an RNA Pseudoknot. 1. Mol. Bioi. 214:437-453.
RajBhandary V.L., Stuart A., Faulkner R.D., Chang S.H., and Khorana H.G.(l966). Nucleotide Sequence Studies on
Yeast Phenylalanine sRNA. pp. 425-434.Volume XXXI Cold Spring Harbor Symposia on Quantitative Biology
Rinke-Appel J., Junke N., Brimacombe R., Lavrik I., Dokudovskaya S., Dontsova 0., Bogdanov A. (1994). Contacts
between 16S ribosomal RNA and mRNA. within the spacer regon separating the AUG initiator codon and the Shine·
Oalgrano sequence; a site-directed cross-linking study. Nucleic Acids Res. 22:3018-3025.
Rosendahl G., Hansen L.B., and Douthwaite S. (1995). Pseudoknot in domain II of 23S rRNA is essential for ribosomal
function. 1. Mol. Bioi. 249:59-60.
SantaLucia J., Kierzek R., Turner D.H. (1992). Context dependence of Hydrogen bond free energy revealed by substi-
mtions in an RNA hairpin. Science 256:217-219.
Sirum-Connolly K. and Mason T.L. (1993). Functional requirement of a site-specific ribose methylation in ribosomal
RNA. Science 262:1886-1889.
Stiege W., Glotz C., and Brimacombe R. (1983). Localisation of a series of intra-RNA cross-links in the secondary and
tertiary structure of 23S RNA, induced by ultraviolet irradiation of Escherichia coli 50S ribosomal subunits. Nucleic
Acids Res. 11:1687-1706.
Stiegler P., Carbon P., Ebel J.P., Ehresmann C. (1981). A General Secondary-Structure Model for Procaryotic and
Eucaryotic RNAs of the Small Ribosomal Subunits. Eur. 1. Biochem. 120:487-495.
Szewczak A.A., Moore P.B., Chan Y~L., and Wool I.G. (1993). The confonnation of the sarcinlricin loop from 28S
ribosomal RNA. Proc. Natl. Acad. Sci. USA 90:9581-9585.
Trust TJ., Logan S.M., Gustafson C.E., Romaniuk PJ., Kim N.W., Chan V.L., Ragan M.A.• Guerry P., and Gutell
R.R. (1994). Phylogenetic and Molecular Characterization of a 23S rRNA gene positions the genus Campylobacter
in the epsilon subdivision of the Proteobacteria and shows that the presence of transcribed spacers is common in
Campylobaeter spp. 1. Bacteriology 176: 4597-4609.
ten Dam E., Pleij K., and Draper D. (1992). Structural and Functional Aspects of RNA Pseudoknots. Biochemistry
31 :11665-11676.
Tuerk C., Gauss P., Thermes C., Groebe D.R., Gayle M., Guild N., Stonno G., D'Aubenton-Carafa Y., Uhlenbeck
O.C., Tinoco I., Brody E.N., and Gold L. (1988). CUUCGG hairpins: Extraordinarily stable RNA secondary
structures associated with various biochemical processes. Proc. NtJtl. Acad. Sci. USA 85:1364-1368.
Turner D.H., Sugimoto N., aod Freier SM. (1988). RNA Structure Prediction. Annu. Rev. Biophys. Biophys. Chem.
17:167-192.
Varani G., Cheong C., and Tinoco I. Jr. (1991). Structure of an Unusually Stable RNA Hairpin. Biochemistry 30:3280-
3289.
Vila A., Viril-Farley J., and Tapprich W.E. (1991). Pseudoknot in the central domain of small subunit ribosomal RNA
is essential for translation. Proc. Narl. Aead. Sci. USA 91: 11148-11152.
Winker, S. and Woese, C. R. (1991) A Definition of the Domains Archaea. Bacteria and Eucarya in Tenus of Small Subunit
Ribosomal RNA Characteristics. System. Appl. Microbiol. 14:305-310
Woese C.R.• Magnun LJ., Gupta R., Siegel R.B., Stahl D.A., Kop J., Crawford N., Brosius J., Gutell R., HoganJ,J.,
and Noller H.F. (1980). Secondary structure model for bacteriall6S ribosomal RNA: phylogenetic. enzymatic and
chemical evidence. Nucleic Acids Res. 8:2275-2293.
Woese C.R. (1980). Just So Stories and Rube Goldberg Machines: Speculations on the Origin of the Protein Synthetic
Machinery. In Ribosomu: Structure. Function. and Genetics. Chambliss G.. Craven G.R.. Davies J.• Kahan L.. and
Nomura M., eds. University Park Press. Baltimore.

18. -
•
128
Woese C.R., Gulell R.. Gupta R., Noller H.F. (1983). Detailed analysis of the higher-order structure of 16S-like ribosomal
ribonucleic acids. Microbial. Rev. 47:621-669.
Woese C.R. and Gutell R.R. (1989). Evidence for several higher order structural elements in ribosomal RNA. Proc. Natl.
Acad. Sci. USA 86:31 19-3122
Woese, C.R., Winker S., and Gulell R.R. (1990). Architecture of Ribosomal RNA: Constraints on the sequence of Tetra-
loops. Proc. NaIf. Acad. Sci. USA 87:8467-8471.
Woese, C.R. (1987). Bacterial Evolution. Microbiological Reviews 51:221-271.
Woese. C.R. and Pace R. N. (1993). Probing RNA Structure. Function. and History by Comparative Analysis. p.91-117.
In The RNA World, Cold Spring Harbor Laboratory Press.
Yonath A., Bennett W., Weinstein S., and Wittmann H.G. (1990). Crys14l1ography and Image Reconstuc(ions of
Ribosomes. pp.134-147. In The Ribosome: Structure. Function & Evolution. Hill et aI., eds. American Society for
Microbiology.
Zachau H.G., Dutting D.• Feldmann H., Melchers F., and Karau W. (1966). Serine Specific Transfer Ribonucleic Acids.
XIV. Comparison of Nucleotide Sequences and Secondary Structure Models. pp. 417-424. Volume XXXI. Cold
Spring Harbor Symposia on Quantilalive Biology.
Zwieb C., Glatz C., and Brimacombe R. (198 I). Secondary structure comparisons between small subunit ribosomal RNA
molecules from six different species. Nucleic Acids Res. 9:3621-3640.

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Ribosomal RNA
Structure, Evolution,
Processing, and Function
in Protein Biosynthesis
Edited by
Robert A. Zimmermann
Albert E. Dahlberg
CRCPress
Boca Raton New York London Tokyo

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Library of Congress Cotaloging.in.PubUClltloD Dolo
Ribosomal RNA : structure. evolution. processing. and function in prolein biosynthesis I Roben A. Zimmennann.
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ISBN 0-8493·8864-3
i . RNA. 2. Ribosomes. I. Zimmennann. Roben A. II. Dahlberg. Alben E.
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