Lee J.C., Gutell R.R., and Russell R. (2006). The UAA/GAN internal loop motif: a new RNA structural element that forms a cross-strand AAA stack and long-range tertiary interactions. Journal of Molecular Biology, 360(5):978-988.

1. The UAA/GAN Internal Loop Motif: A New RNA
Structural Element that Forms a Cross-strand AAA
Stack and Long-range Tertiary Interactions
Jung C. Lee1,2
, Robin R. Gutell1,2
⁎ and Rick Russell1,3
⁎
1
The Institute for Cellular and
Molecular Biology,
The University of Texas at
Austin, 1 University Station
A4800, Austin,
TX 78712-0159, USA
2
Section of Integrative Biology,
The University of Texas at
Austin, 1 University Station
A4800, Austin,
TX 78712-0159, USA
3
Department of Chemistry and
Biochemistry, The University of
Texas at Austin, 1 University
Station A4800, Austin,
TX 78712-0159, USA
Analysis of aligned RNA sequences and high-resolution crystal structures
has revealed a new RNA structural element, termed the UAA/GAN motif.
Found in internal loops of the 23 S rRNA, as well as in RNase P RNA and
group I and II introns, this six-nucleotide motif adopts a distinctive local
structure that includes two base-pairs with non-canonical conformations
and three conserved adenine bases, which form a cross-strand AAA stack in
the minor groove. Most importantly, the motif invariably forms long-range
tertiary contacts, as the AAA stack typically forms A-minor interactions and
the flipped-out N nucleotide forms additional contacts that are specific to
the structural context of each loop. The widespread presence of this motif
and its propensity to form long-range contacts suggest that it plays a critical
role in defining the architectures of structured RNAs.
© 2006 Elsevier Ltd. All rights reserved.
*Corresponding authors
Keywords: RNA structural motif; long-range RNA tertiary interaction;
comparative sequence analysis; base pair conformation; evolution
Introduction
The higher-order structure of RNA is composed of
a set of helices and loops that are connected by
tertiary interactions to form the three-dimensional
structure. Many of these tertiary interactions are
formed by structural “motifs”, modules of structure
that repeat in multiple places and in multiple
RNAs.1 Thus, identifying and characterizing these
motifs and their contact partners are crucial for
understanding and ultimately predicting higher-
order RNA structures.
Two types of analysis provide powerful and com-
plementary information in defining structural motifs
and contacts. Comparative sequence analysis, which
identifies similar structures in a set of functionally
equivalent RNA sequences, accurately predicted
nearly all of the base-pairs within the 16 S and 23 S
rRNA secondary structures,2–4 and it has been used
to identify biases in the distribution of nucleotides
within structured RNAs and to delineate several
motifs and tertiary contacts.5–10 One of the most
pronounced biases is the preponderance of adeno-
sine nucleotides that are unpaired in the secondary
structure. In these rRNAs, as well as in the group I
introns, the ratio of unpaired to paired adenosine
bases is nearly two, whereas analogous ratios for the
other three nucleotides are much less than one.11,12
Consistent with this bias, several of the motifs that
have been identified in unpaired regions of the
secondary structure have a high frequency of
adenosine bases.5,7,9,10,13 A second approach has
been introduced more recently by the solution of
high-resolution crystal structures of large RNAs.
These structures, which provide snapshots of RNAs
and the conformations of their components with
unparalleled precision, have been used to identify
and characterize several structural motifs,14–16 as
well as to classify the tremendous diversity of base-
pair conformations found in structured RNAs.17
Although the two types of analysis have been
used independently with great success, they can be
even more powerful when combined. Crystal
structures can provide high-resolution views of the
motifs and contacts within an RNA but dynamics of
the molecule are not visible, nor is it possible to
determine the relative energetic contributions of the
E-mail addresses of the corresponding authors:
robin.gutell@mail.utexas.edu; rick_russell@mail.utexas.edu
doi:10.1016/j.jmb.2006.05.066 J. Mol. Biol. (2006) 360, 978–988
0022-2836/$ - see front matter © 2006 Elsevier Ltd. All rights reserved.

2. myriad secondary and tertiary contacts. In contrast,
a limitation of comparative sequence analysis is that
it does not determine the structure directly; instead
it provides sequence conservation and variation
data from which structural features are inferred.
However, a key strength is that additional informa-
tion is encrypted in the patterns of evolutionary
sequence change, which can provide insight into the
relative importance of tertiary contacts within an
RNA, its interactions with other cellular compo-
nents, and its function. Thus, these two types of
analysis are highly complementary and, when
applied together, have broadened and deepened
our understanding of RNA tertiary structure and its
components.10,13,16,18,19
One of the most prominent structural features to
emerge from analysis of sequences and structures is
the A-minor interaction, formed between unpaired
A bases in secondary structure and the minor
groove surface of another duplex.6,16,19 These
interactions are present in virtually all high-resolu-
tion structures of large RNAs,18,20–25 and indeed are
formed by a significant fraction of the unpaired A
bases in secondary structures.16,19 Many A-minor
interactions involve contacts between A bases of
hairpin loops and their receptor duplexes elsewhere
within an RNA, constraining the tertiary structure
by defining the position of the hairpin loop relative
to its receptor.6,10,26 In addition, many A-minor
interactions are formed by adenosine bases within
internal loops, providing further constraints on the
positions and orientations of duplexes within the
higher-order RNA structure.16,19,27
Although many of the unpaired A bases in the 16 S
and 23 S rRNAs are present in motifs that have been
characterized, approximately 350 of them do not fall
in existing motifs.16,17 We have therefore underta-
ken a systematic analysis of comparative sequences
and high-resolution crystal structures to identify
additional motifs involving these unpaired adeno-
sine bases . Here we report the discovery of a new
RNA structural motif with the consensus sequence
UAA/GAN. This motif includes three conserved A
bases that are unpaired in the secondary structure
and positions them in a cross-strand AAA stack,
allowing them to form A-minor tertiary interactions.
Results
Sequence and structural characteristics of the
UAA/GAN motif
An analysis of the 16 S and 23 S rRNA compa-
rative structures and our database of aligned
sequences28 revealed seven internal loops of the
23 S rRNA that include the sequence 5′-UAA on one
strand and 5′-GAN on the other. These loops are
listed by the position numbers of their 5′ most
nucleotides in Table 1, and their locations within the
23 S rRNA secondary structure are shown in Figure
1(a) and (c). Of the seven loops, three are present in
all three phylogenetic domains and conform to the
UAA/GAN consensus sequence in at least two-
thirds of the aligned sequences (Supplementary
Data, Table 1). The remaining four are present in
one or two phylogenetic domains and have varying
degrees of sequence conservation. The N of the motif
is most commonly A or U but can also be G or C.
Interestingly, the consensus motif is absent from the
16 S rRNA sequences, but several additional
examples of the motif are present in a collection of
aligned RNase P RNAs and group I and group II
intron sequences (see below: UAA/GAN motif in
non-ribosomal RNAs).
Of the seven UAA/GAN internal loops within the
23 S rRNA, three are present in the high-resolution
crystal structure of the archaeal 50 S ribosomal
subunit of Haloarcula marismortui,15,18 as well as the
structures from the bacteria Deinococcus radio-
durans29 and Escherichia coli.30 A fourth internal
loop is present in all three structures with a closely-
UAA/GAN loop is present only in the bacterial
structures.29,30 The remaining two loops are identi-
fied as UAA/GAN from comparative sequence
analysis, but they have distinct sequences in H.
marismortui and adopt distinct conformations. All
seven loops and their contacts are shown schema-
tically in Figure 2, and the structure of a represen-
tative loop from each panel of Figure 2 is shown in
the corresponding panel of Figure 3. (Structures of
the remaining loops are shown in Supplementary
Data, Figure 1).
Table 1. The UAA/GAN motifs and their tertiary
contacts mediated by the cross-strand AAA stack
Motif
AAA
stack
AAA
receptor
A-minor
interaction
H23S-1096 (E23S-999) A1098 G1075:C1084 Type I
A1097 G1074:C1085 Type II
A1259 G1074:C1085 Type I
H23S-2774 (E23S-2739) A2776 C2559:G2574 Type I
A2775 G2558:C2575 Type II
A2799 G2558:C2575 Type I
H23S-1457 (E23S-1352) A1459 C783:G863 Type I
A1458 A784:U862 Type II
A1485 A1656 –
H23S-664 (E23S-607) A666 C208:G231 Type I
A665 G209:C230 Type II
A682 G209:C230 Type I
E23S-1418 A1580 – –
A1579 – –
A1419 A1494 –
H23S-1579a
(E23S-1475) A1581 G1540:U1645 Type I
A1580 – –
A1615 G1541:C1644 Type II
A1616 G1542:C1643 Type I
H23S-1908a
(E23S-1852) A1910 U2128:A2265 Type I
A1909 C2127:G2266 Type II
A1930 C2127:G2266 Type II
A1931 C2126:G2267 Type I
The UAA/GAN motifs are named by the H. marismortui 23 S
rRNA (H23S) and their most 5′ nucleotide position with their E.
coli equivalents (E23S) in parentheses, except for E23S-1418,
which is not present as UAA/GAN in H. marismortui.
a
Although these positions have UAA/GAN in many 23 S
rRNAs, related sequences in the H. marismortui 23 S rRNA adopt
an alternative structure including an cross-strand AAAA stack.
979Long-range Interactions with the UAA/GAN Motif

3. Figure 1. Locations of the UAA/GAN and related motifs: (a) H. marismortui 23 S rRNA; (b) T. thermophilus 16 S rRNA;
(c) E. coli 23 S rRNA fragment; (d) B. subtilus RNase P RNA; (e) B. stearothermophilus RNase P RNA. The UAA/GAN motif
and its close relatives are shaded in light green. Other related motifs are: light blue for the UNA/GAN motif in three-way
junctions and pink for the GA/GAA motif in internal loops. The tertiary interactions are shown either in continuous red
lines for base-base H-bonding interactions or in dotted red lines for other types of H-bonding interactions. The letter and
the arrow indicate in which phylogenetic domains the motifs are present: A, archaea; B, bacteria; E, eukaryotes. Letters in
parenthesis indicate that the motif is present in only a small fraction of sequences.
980 Long-range Interactions with the UAA/GAN Motif

4. Detailed analysis of the four UAA/GAN loops
present in the H. marismortui structure (including the
UAA/GGA loop) reveals striking structural char-
acteristics, defining UAA/GAN as a structure motif
as well as a sequence motif. The motif includes two
base-pairs with specific conformations; the U of
UAA and the A of GAN form a base-pair in the
reversed Hoogsteen conformation (U:A rH), and the
final A of UAA and the G of GAN form a sheared
pair (A:G S).17 This latter pair is adjacent to a regular
secondary helix and has previously been identified
as a common motif at helix ends.13 The three
conserved A bases of the motif form a cross-strand
AAA stack in the minor groove, such that they are
available to form tertiary contacts. The middle A of
the stack (the first A of UAA) does not have a base-
pairing partner on the other strand, producing a
significant bend in the helix (40°−60°; Figure 4). As
described below, this bend increases the exposure of
the stacked adenine bases for tertiary contact
formation by broadening the minor groove. A
final feature of the local motif structure is that
the N of GAN is not involved in base-pairing within
the loop and is flipped out of the helix, where it is
also available to form tertiary contacts. Pair-wise
comparisons of the four UAA/GAN loops in the H.
marismortui structure show that their local structures
are nearly identical, with RMSD values ranging
from 2 Å to 3 Å (Supplementary Data, Figure 2(a)
and (b)). In contrast, analogous comparisons with
structures of related motifs8,12,31–34 give substantial-
ly larger RMSD values (Supplementary Data, Figure
2(c) and (d)), demonstrating that the full comple-
ment of structural features described above is
unique to the UAA/GAN motif.
A-minor tertiary interactions of the AAA stack
As noted above, the base-pairing pattern and
formation of the cross-strand AAA stack result in a
broadening of the minor groove and narrowing of
the major groove within the loop. The broadened
minor groove increases the exposure of the A
bases in the stack, facilitating formation of A-
minor tertiary contacts between these bases and
double-stranded “receptor” regions. Indeed, all
four UAA/GAN loops in the H. marismortui
structure form A-minor interactions (Table 1).
Most of the receptors are remote in the secondary
structure (Figure 1(a)), and thus the motif plays an
important role in defining the higher-order struc-
ture of the 23 S rRNA.
There is also strong conservation of several
molecular features of the tertiary interactions. Three
of the four UAA/GAN loops in the H. marismortui
structure have essentially identical patterns of
tertiary contacts formed by the AAA stack, and the
Figure 2. The UAA/GAN internal loops of the 23 S
rRNA and their tertiary contacts. (a) Schematic descrip-
tion of the canonical motif, with the consensus
UAA/GAN sequence, and examples from simple six-
nucleotide internal loops; (b) a loop that has the
consensus UAA/GAN sequence and additional un-
paired nucleotides 3′ of GAN; (c) a variant of the motif
with the UAA/GGA sequence; and (d) non-canonical
loops of the H. marismortui 23 S rRNA that contain
UAA/GAN sequences in other organisms. Nucleotides
that form tertiary contacts are indicated in the
schematic diagrams with asterisks (*), nucleotides that
are flipped out of the helix are indicated with zig-
zagged arrows, and the A bases that form the cross-
strand AAA stack are blocked together using dotted
lines. Their tertiary contacts are shown similarly as in
Figure 1. Nucleotides are numbered according to the H.
marismortui 23 S rRNA (H23S), except for loop
E23S-1418, which is not present in H. marismortui and
is numbered according to the E. coli 23 S rRNA (E23S).
The structure and tertiary contacts of a representative
motif from each panel (the one with an asterisk) are
shown in the corresponding panel of Figure 3, and the
rest (the ones marked with a double asterisk) are
shown in Supplementary Data, Figure 1.
981Long-range Interactions with the UAA/GAN Motif

5. fourth has the same pattern for two of the three A
bases. The interactions of one of the three similar
loops are shown in Figure 3 and the rest are shown in
Supplementary Data, Figure 3. The A of the A:G base-
pair (UAA) forms a canonical type I A-minor
interaction with a highly conserved C:G or G:C
Figure 3. Stereo views of the structure and tertiary contacts of the UAA/GAN motif. (a) H23S-1096 (domain II). The
AAA stack (A1098-A1097-A1259) interacts with two consecutive base-pairs, G1074:C1085 and G1075:C1084, and the N of
GAN (G1260) is flipped out and contacts A1088. (b) H23S-1457 (domain III). The interactions of the AAA stack
(A1459-A1458-A1485) are idiosyncratic, as two of the As in the AAA stack (A1459 and A1458) form A-minor interactions
with two consecutive base-pairs in a helix of domain II (C783:G863 and A784:U862), while the third A (A1485) forms a
reversed wobble pair with A1656, from another region of domain III (A:A rWb).17
Interactions of the N of GAN (A1486) and
the adjacent nucleotides (A1487 and U1488) are shown here and described in the text. (c) H23S-664 (domain II), which has
the non-canonical sequence UAA/GGA but forms a conformation and tertiary contacts that are analogous to those of the
canonical motif. The AAA stack (A666-A665-A682) interacts with two consecutive base-pairs in domain I, C208:G231 and
G209:C230. The second G of GGA (G681) is moved into the major groove and stacked onto U664, so that it does not interfere
with the AAA stack. (d) H23S-1579 (domain III). The AAAA stack (A1581-A1580-A1615-A1616) interacts with three
consecutive base-pairs in the same domain. For each panel (a–d), a quasi-3D schematic diagram of the motif (left panel) is
shown in the same orientation as in the stereo Figure, with the AAA or AAAA stack blocked with thin black dotted lines.
982 Long-range Interactions with the UAA/GAN Motif

6. base-pair that is part of a continuous helix (Table 1
and Supplementary Data, Table 2). In two of the three
UAA/GAN loops (H23S-1096 and H23S-664), the
adenine base is on the G side of the receptor base pair,
as described previously for the canonical type I
interaction,16,19 forming a reversed sheared confor-
mation (G:A rS).17 In contrast, in the third example of
the motif (H23S-2774), the adenine base is on the C
side, forming a reversed parallel sheared conforma-
tion (G:A rpS). Even in this case, the adenine base is
likely to be on the G side and form a canonical type I
interaction in most rRNAs, as the vast majority of 23 S
sequences have the receptor base-pair reversed
relative to the H. marismortui 23 S rRNA (Supple-
mentary Data, Table 2). The next A in the stack, the
unpaired A of UAA, forms a type II A-minor inter-
action with a receptor base-pair that is adjacent to the
one contacted by the first A. The final A of the stack,
the A of GAN, interacts with this same base-pair as
the middle A, forming a variant type I interaction.
Thus, a distinctive feature of the UAA/GAN motif is
that the AAA stack forms a characteristic set of
A-minor interactions with only two consecutive
base-pairs, in contrast to other motifs in which
stacked A bases form A-minor interactions with the
equivalent number of base-pairs.16,19,20,35 Because
the latter two A bases are significantly propeller-
twisted relative to the plane of the receptor base-pair,
each of them is unable to interact with both
nucleotides of the pair, and the final A does not
achieve the full network of canonical type I interac-
tions (Figure 5). Perhaps because of the more limited
nature of this interaction, the identity of its receptor
base-pair is much less conserved than the other
receptor base-pair (Supplementary Data, Table 2).
Additional tertiary contacts of the UAA/GAN
motif and flanking nucleotides
In each of the canonical UAA/GAN loops in the H.
marismortui crystal structure, the N of GAN is ex-
truded from the helix and forms a tertiary contact
(Figure 3(a) and (b); Supplementary Data, Figure
1(a)). In loop H23S-1096 (Figure 3(a)), the N nucle-
otide (G1260) forms a base-triple with A1088:G1072,
whereas in H23S-2774 (Supplementary Data, Figure
Figure 4. Overall helix bending produced by the UAA/GAN motif and its close relatives. The A bases of the AAA or
AAAA stack are shown in red and labeled with their position numbers in the H. marismortui 23 S rRNA.
983Long-range Interactions with the UAA/GAN Motif

7. 1(a)), the N nucleotide (A2800) forms a parallel
flipped sheared base-pair with A2576 (A:A pfS).17 In
each of these loops, the N contacts a nucleotide that is
a neighbor in primary sequence of the receptor for
the AAA stack, and the additional interaction pre-
sumably functions to strengthen the tertiary contact
of the AAA stack. In the third loop (H23S-1457),
however, the N nucleotide (A1486) forms a variant
A-minor interaction with G1452:C1675, which is far
removed in the secondary structure from the
receptor for the AAA stack (Figure 3(b)).
The H23S-1457 loop is also unusual in that there
are two additional bulged nucleotides that immedi-
ately follow the N of GAN (Figures 2(b) and 3(b)),
each of which is extruded from the helix and inter-
acts with a different region of the rRNA. The first
base, A1487, forms an A-minor interaction with
U1412:G1697, in a distinct region of domain III. The
second base, U1488, hydrogen bonds with the 2′-OH
of G1697 (the same nucleotide contacted by A1487
above) and forms a novel “U-minor” interaction
with U785:A861, which is in domain II and adjacent
to the receptor of the AAA stack. Together, the in-
teractions of this internal loop bring five regions of
the rRNA from domains II and III into proximity
(Figure 1(a)). These additional bulged nucleotides
are present in archaeal and eukaryotic sequences but
absent from bacterial sequences. Interestingly, the
same regions also come together in the bacterial
crystal structures,29,30 indicating that the additional
bulged nucleotides are not the only possible strategy
for assembling these domains. It is not clear whether
the alternative strategies that must be employed by
the bacterial 50 S subunit are also used by the
archaeal counterpart in addition to the network of
contacts formed by the UAA/GAN motif and its
flanking nucleotides.
Evolutionary interchanges of UAA/GAN and
related motifs
There are several examples of the UAA/GAN
motif exchanging through evolution with variants
of the motif. As noted above, the H23S-1457 loop
includes the extra unpaired nucleotides only in
archaeal and eukaryotic sequences but maintains
the UAA/GAN consensus sequence across all
three phylogenetic domains (Supplementary Data,
Table 1). A second loop, H23S-664, has the
sequence UAA/GGA in H. marismortui and adopts
a conformation that is essentially identical to that
of the canonical UAA/GAN motif despite the
sequence difference (Figures 2(c) and 3(c)). The
second G of GGA represents an insertion that has
minimal effect on the overall structure. It is not
paired, but instead retreats into the major groove
and stacks onto the U of the U:A base-pair. This
conformation allows formation of an AAA stack in
the minor groove, a bend in the compound helix
(Figure 4), and formation of A-minor tertiary
contacts (Supplementary Data, Figure 3), all of
which are indistinguishable from those of the
canonical motif. Thus, this UAA/GGA sequence
is most appropriately considered a variant of the
UAA/GAN motif rather than a distinct motif. The
loop has apparently changed between the canon-
ical and variant sequences multiple times through
evolution, as both versions of the motif are present
in significant fractions of archaeal and bacterial
sequences (Supplementary Data, Table 1).
There are also examples of exchanges between
UAA/GAN and related but distinct motifs
(H23S-1579 and H23S-1908; Figures 1(a) and 2(d)).
Both of these internal loops are UAA/GAN in
significant fractions of aligned sequences, but they
have non-canonical sequences in H. marismortui and
adopt related but distinct structures. The H23S-1579
loop is UAA/GAA in a modest fraction of archaeal
sequences but is CAA/GAA in H. marismortui
(Supplementary Data, Tables 1 and 2). It forms two
base-pairs that are analogous to those of the
canonical UAA/GAN motif, with a C:A reversed
Hoogsteen pair replacing the U:A pair of the
canonical motif. The H23S-1908 loop is UAA/GAN
in most of the bacterial sequences, but it is
Figure 5. A-minor tertiary interactions mediated by
the cross-strand AAA stack of the UAA/GAN motif
(H23S-1096). The top panel shows A1098 forming a
canonical type I interaction. The middle and bottom
panels show A1097 and A1259 forming type II and
variant type I interactions, respectively, with the base-
pair adjacent to that contacted by A1098. Because there
is substantial propeller twist between the AAA stack
and the receptor base-pairs, A1259 interacts only with
G1074, whereas an additional contact with the 2′-OH of
C1085 would be present in a canonical type I
interaction.16
The A-minor tertiary interactions mediated
by other UAA/GAN loops of the 23 S rRNA are shown
in Supplementary Data, Figure 3.
984 Long-range Interactions with the UAA/GAN Motif

8. UAA/GAN in only a small fraction of archaeal
sequences and is GAA/GAA in H. marismortui and
has a distinct base-pairing pattern in the crystal
structure (Supplementary Data, Figure 1(b)).
Despite the differences in base-pairing, these
internal loops have structural features in common
with each other and with the canonical UAA/GAN
motif. Both loops have unpaired A bases that form a
cross-strand stack in the minor groove, but in each
loop there are four stacked A bases instead of three
(Figure 3(d)). Also similar to the UAA/GAN motif,
these loops include significant bending of the helix;
the bending angle for loop H23S-1579 is in the range
of the canonical UAA/GAN motif (Figure 4),
whereas that for loop H23S-1908 is decreased to
∼25°, which may be due, at least in part, to a C:C
mis-pair adjacent to the motif sequence. Addition-
ally, both AAAA stacks form A-minor tertiary
contacts, but these contacts are with three consecu-
tive receptor base-pairs instead of two. Despite the
sequence and structure differences between these
loops and the canonical UAA/GAN motif, they
appear to function similarly, generating cross-strand
stacks of A bases that form A-minor tertiary
contacts.
Further highlighting the ability of these loops to
switch between related motifs, both of them have
variations in E. coli relative to H. marismortui. The
H23S-1908 equivalent in E. coli has the canonical
sequence UAA/GAA, yet adopts the AAAA stack
described above and forms the same tertiary
contact as the H23S-1908 loop in H. marismortui,
suggesting that this alternative structure is im-
posed by the tertiary contacts formed by this loop
(see Discussion). The H23S-1579 equivalent in E.
coli has GUA/GAG and adopts a conformation
that is similar to the E-like loop motif and distinctly
different from the UAA/GAN motif.30,33 The two
outer A bases are stacked on a central U:A reversed
Hoogsteen pair, which is moved into the minor
groove such that this AAA stack is in the minor
groove. This internal loop retains both the charac-
teristic helix bend of the UAA/GAN motif and the
characteristic tertiary contact formation. In addition
to a new contact, a Watson–Crick pair with a
nucleotide from domain IV, the bacterial motif
retains the intra-domain contact within domain III
that is formed by the H23S-1579 loop in H.
marismortui (Figure 1(c)).
UAA/GAN motif in non-ribosomal RNAs
In contrast to the relatively large number of
UAA/GAN internal loops in the 23 S rRNA, there
are no examples of the motif in our database of
aligned 16 S rRNA sequences. On the other hand,
several examples are present in other RNAs.
Analysis of RNase P RNA sequence alignments36
revealed two examples in the type B RNase P RNAs,
in L8 and P15.1 (Figure 1(d) and (e)). Interestingly,
the UAA/GAN loop in L8 is part of a ten-nucleotide
hairpin loop in the secondary structure. In the
crystal structure of the specificity domain from B.
subtilis, this hairpin loop includes the canonical
UAA/GAN motif structure, with the characteristic
two base-pairs and the AAA stack, which is flanked
by a tetraloop.37 Further, in the crystal structure of
the intact RNase P RNA from B. stearothermophilus,
this sequence forms a long-range tertiary connection
to the catalytic domain.24 Interestingly, in this
structure the UAA/GAN internal loop does not
form the U:A rH base-pair and forms an AAAA
stack rather than the canonical AAA stack, interact-
ing with three consecutive base-pairs in P4. The
internal loop structure and tertiary contacts are
similar to those of the H23S-1908 loop, described
above, further suggesting that the UAA/GAN motif
is capable of rearranging to an alternative confor-
mation upon tertiary contact formation. The P15.1
internal loop has the sequence UAA/GAG in B.
subtilis, but is present as GAA/GGA, a distinct
motif, in the crystal structure from B. stearo-
thermophilus.24 The loop gives a similar AAA stack
in the minor groove by forming three consecutive
sheared G:A base-pairs (G:A S) and forms type II
A-minor tertiary contacts with P15.
We also identified the UAA/GAN motif by
sequence analysis in group I and group II self-
splicing introns. In some group I introns, the motif is
present in the J4/5 region (data not shown). The
corresponding internal loop in the Azoarcus Ile-tRNA
group I RNA crystal structure has a different
sequence, AAA/CAA, but forms a similar cross-
strand AAA stack in the minor groove, which forms
A-minor tertiary contacts with two consecutive base-
pairs at the 5′-splice site.23 In some group II introns,
an internal loop in the helix between subdomains IA
and ID contains the UAA/GAN motif sequence
(data not shown). It is not yet known whether this
internal loop participates in a tertiary contact.
Discussion
We have used sequence and structure analysis to
identify UAA/GAN as an internal loop motif that
forms a characteristic structure and mediates a
characteristic set of long-range tertiary interactions.
The motif is present in seven internal loops of the 23 S
rRNA, and most of these loops form tertiary contacts
that connect different secondary structure domains.
Interestingly, the motif is absent from the 16 S rRNA,
which may be a consequence of the relatively low
density of long-range tertiary contacts in the 16 S
rRNA compared with the 23 S rRNA.4,18,22 How-
ever, it is also present in the type B RNase P RNAs,
where it mediates the only inter-domain connection
identified in the crystal structure.24
Central structural features of the UAA/GAN
motif are a cross-strand stack of three A bases and
the formation of long-range tertiary contacts by
docking of these bases into A-minor contacts with a
receptor helix. A-minor contacts have been demon-
strated to be widespread elements of RNA structure
and are associated with several structural motifs.16
There is a rapidly expanding family of internal loop
motifs that generate cross-strand purine stacks,
985Long-range Interactions with the UAA/GAN Motif

9. including the E-loop,31 GGA/GAA and a family of
related motifs,33,34 and the tandem G:A motif.38–40
Although these other motifs are related to the
UAA/GAN motif, they do not produce the helix
bend and often do not form tertiary contacts. There
are also several more distantly related but function-
ally similar motifs in that they generate intra or inter-
strand purine stacks and form A-minor tertiary
contacts, including GNRA tetraloops6,20,41,42 and
lone-pair triloops.10 Another interesting example is
the K-turn,15 which typically includes a purine stack
that makes its A-minor contact locally instead of
long-range, producing a sharp bend in the com-
pound helix.
In addition to these families, our sequence and
structure analysis revealed two new motifs that are
quite closely related to the UAA/GAN internal loop
motif. First is the GA/GAA motif, which, like the
UAA/GAN motif, produces helical bending and
forms an AAA stack that is commonly involved in A-
minor tertiary interactions (Figure 1). Although the
global structures and functional roles of the two
motifs are similar, they are distinct in their sequence
and base-pairing characteristics. Second is a motif
within three-way junctions that has essentially the
same sequence, UNA/GAN, but does not have the
same structural features (Figure 1(a) and (b)). Instead
of forming an AAA stack and generating a helical
bend, the three-helix junction motif flips out the N of
UNA and mediates coaxial stacking of the two helices
flanking the UNA sequence (unpublished results).
Like many RNAs, the 23 S rRNA includes regions
that can be considered “peripheral elements”, which
lie toward the outside of the structure and confer
stability by forming connections to each other and to
the structural elements within the core. It is striking
that all seven examples of the UAA/GAN motif are
situated near the surface of the 50 S structure, where
they mediate tertiary contacts between different
domains. Other motifs that commonly form long-
range A-minor tertiary interactions, notably the
GA/GAA, GNRA tetraloop, and lone-pair triloop
motifs, also occur most often near the surface of the
50 S structure. Together, these motifs appear to play
a fundamental role in defining the spatial relation-
ships of the domains as they form the global archi-
tecture of the native 50 S subunit.
In addition to forming stable tertiary contacts, it is
possible that some A-minor contacts formed by the
UAA/GAN and related motifs are formed dynami-
cally during translation. Transient A-minor interac-
tions have been demonstrated to play a role in
recognition of tRNAs during translation43,44 and are
implicated in ribosomal translocation.45 Further, A-
minor interactions have been proposed as attractive
candidates for dynamics more generally because they
are expected to form and break rapidly enough to be
compatible with biological processes and because the
contact partners are typically able to maintain their
local conformations while the contact is broken.45
Consistent with the possibility of dynamic interac-
tions by the UAA/GAN motif, the E23S-1418 loop,
which does not form A-minor tertiary contacts in the
E. coli crystal structure, adopts essentially the same
conformation as other UAA/GAN-containing loops,
indicating that the UAA/GAN motif can retain its
local structure in the absence of its tertiary contacts
(Supplementary Data, Figure 1C). On the other hand,
the UAA/GAN motif can apparently rearrange upon
formation of a contact, as L8 of RNase P RNA and the
E23S-1908 internal loop adopt conformations that
include four stacked A bases, instead of three, when
their tertiary contacts are formed (see Results). The
ability of RNA elements to adopt multiple conforma-
tions, which has been documented,46,47 could be a
benefit for dynamic interactions under some circum-
stances, because it could permit the same sequence to
adjust its contact-forming surface and thus to interact
with multiple and varied contact partners during the
course of a reaction.
Our sequence analysis revealed several examples
of evolutionary interchanges between UAA/GAN
and related motifs. Some changes are conservative,
with a motif being replaced by a related motif that
makes a similar tertiary contact, whereas others are
quite non-conservative. Several conservative
changes are described in Results (H23S-1457, H23S-
1579, H23S-1908) where UAA/GAN is replaced by a
sequence that also forms a cross-strand purine stack
and forms essentially the same tertiary contact.
There are also more radical evolutionary inter-
changes. The E23S-1418 loop interacts with a hairpin
loop (Figure 1(c)), but in H. marismortui, which does
not have UAA/GAN at the corresponding position,
the hairpin loop instead interacts with a helix that is
adjacent and 15 base-pairs away from the
UAA/GAN loop in the E. coli rRNA. In another
example, the UAA/GAN sequence within L8 of type
B RNase P RNAs is absent from type A RNase P
RNAs, but the corresponding L8 loop contains a
GNRA tetraloop-like structure, which forms essen-
tially the same inter-domain tertiary contact with P4.
The ability of structured RNAs to exchange be-
tween these motifs during evolution, while retaining
the same global structure and function, underscores
the relatedness of the different motifs as well as
highlighting the modular nature of RNA structure
formation. On the other hand, there are clearly limits
to the exchanges that can be achieved with retention
of function, as some of the UAA/GAN loops within
the 23 S rRNA have the same sequence in the vast
majority of aligned sequences from all three domains
of life (see Supplementary Data, Table 1). Although
the high conservation of some internal loops could
reflect additional functional constraints, their loca-
tions far from the active sites suggest that their prin-
cipal functions are structural. A more likely
possibility is that the changes in tertiary contact
strength or preferred orientations of the contact
partners that presumably arise from exchanging one
motif with another can be accommodated at some
positions within the rRNA more easily than at
others. Indeed, as noted above, there are some
positions in the rRNAs, as well as within RNase P
RNAs and tRNAs,48 where a tertiary contact or motif
can be eliminated completely and replaced with an
986 Long-range Interactions with the UAA/GAN Motif

10. alternative contact. Still, there may be other positions
that have stringent requirements for a particular
motif, either to stabilize the functional structure or
perhaps a critical folding intermediate, or to allow
the interaction to break transiently during dynamic
rearrangements. Addressing these questions will
require a deeper knowledge of the local structures
and energetics of these RNA structural motifs and
their tertiary contacts.
Methods
Identification and characterization of the UAA/GAN
motif in RNAs
From an exhaustive visual mapping of the high-
resolution crystal structures,15,18,22,29,30
we determined
helical base-pairs, long-range tertiary contacts, and other
interactions associated with previously known motifs.4,17
Our understanding of these features in RNA structure
were enhanced with an analysis of our comparative
sequence and structure data.28
Subsequently, the seven
examples of the UAA/GAN motif in the 23 S rRNA and
two examples in the type B RNase P RNA were identified
using similar analysis of the crystal structures and
comparative data. The fragments containing the motifs
and their tertiary contact partners were then excised from
the crystal structures, followed by performing structural
characterization using the RasMol program.49,50
The
UAA/GAN motif was also identified by analysis of the
comparative structure models in group I and II introns.28
Other related motifs were also identified, including the
UNA/GAN and GA/GAA motifs in three-way junctions
and internal loops, respectively. A collection of RasMol
scripts for viewing the structural details for these motifs
and their tertiary interactions are available online†.
Structural comparisons of the UAA/GAN motif and
other RNA structural motifs
To assess the structural relatedness of different UAA/
GAN loops and differences between this motif and two
previously known RNA structural motifs that form a
similar cross-strand AAA stack in the minor groove (the
GGA/GAA and E-loop motifs), a Curves analysis51,52
was
carried out to determine the overall helix bending angles
between the two helices flanking each motif, followed by
RMSD calculations by explicitly superimposing the back-
bone atoms in the two flanking helices in two different
RNA structural motifs using the LSQMAN program.53
Acknowledgements
We thank Whitney Yin for advice on RMSD
calculations. This work was supported by the
National Institutes of Health (GM067317 to R.G
and GM070456 to R.R.) and the Welch Foundation
(F-1427 to R.G. and F-1563 to R.R).
Supplementary Data
Supplementary data associated with this article
can be found, in the online version, at doi:10.1016/
j.jmb.2006.05.066
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Edited by D. E. Draper
(Received 1 April 2006; received in revised form 24 May 2006; accepted 29 May 2006)
Available online 16 June 2006
988 Long-range Interactions with the UAA/GAN Motif