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DomainIIIoftheT. thermophilus 23S rRNA folds
independently to a near-native state
SHREYAS S. ATHAVALE,
1
J. JARED GOSSETT,
1
CHIAOLONG HSIAO,
2
JESSICA C. BOWMAN,
2
ERIC O’NEILL,
2
ELI HERSHKOVITZ,
2
THANAWADEE PREEPREM,
1
NICHOLAS V. HUD,
2
ROGER M. WARTELL,
1
STEPHEN C. HARVEY,
1,2
and LOREN DEAN WILLIAMS
2,3
1
School of Biology and
2
School of Chemistry and Biochemistry, Georgia Institute of Technology, Atlanta, Georgia 30332, USA
ABSTRACT
The three-dimensional structure of the ribosomal large subunit (LSU) reveals a single morphological element, although the 23S
rRNA is contained in six secondary structure domains. Based upon maps of inter- and intra-domain interactions and proposed
evolutionary pathways of development, we hypothesize that Domain III is a truly independent structural domain of the LSU.
Domain III is primarily stabilized by intra-domain interactions, negligibly perturbed by inter-domain interactions, and is not
penetrated by ribosomal proteins or other rRNA. We have probed the structure of Domain III rRNA alone and when contained
within the intact 23S rRNA using SHAPE (selective 29-hydroxyl acylation analyzed by primer extension), in the absence and
presence of magnesium. The combined results support the hypothesis that Domain III alone folds to a near-native state with
secondary structure, intra-domain tertiary interactions, and inter-domain interactions that are independent of whether or not it
is embedded in the intact 23S rRNA or within the LSU. The data presented support previous suggestions that Domain III was
added relatively late in ribosomal evolution.
Keywords: ribosome; large subunit; independent fold; ribosomal evolution; SHAPE
INTRODUCTION
The ribosome is our most direct macromolecular connection
to the distant evolutionary past and to early life (Woese
2001; Wolf and Koonin 2007; Smith et al. 2008; Bokov and
Steinberg 2009; Hsiao et al. 2009; Belousoff et al. 2010; Fox
2010). The ribosome is believed to have emerged from the
‘‘RNA world’’ (Rich 1962; Woese 1967; Crick 1968; Orgel
1968; Gilbert 1986) following an evolutionary pathway that
preserved ribosomal RNAs as central players in peptide
bond formation and decoding (Noller et al. 1992; Ban et al.
2000; Nissen et al. 2000; Harms et al. 2001; Ogle et al. 2001;
Yusupov et al. 2001; Schuwirth et al. 2005; Selmer et al.
2006). Understanding the origin and evolution of rRNA
is a key to understanding the early evolution of life on
earth.
The ribosome is made of a small subunit (SSU) and a
large subunit (LSU). The SSU in bacteria and archaea con-
tains a single RNA molecule, the 16S rRNA. Phylogenetic
studies by Woese et al. (1980) revealed three major and one
minor secondary structural domains (2°domains) of the
16S rRNA. These 2°domains are segregated into indepen-
dent and autonomous three-dimensional domains (3D do-
mains) in the assembled SSU. Each 2°domain of the 16S
rRNA folds and assembles with the appropriate ribosomal
proteins into a 3D domain, independent of other 2°do-
mains (Weitzmann et al. 1993; Samaha et al. 1994; Agalarov
et al. 1999). One 3D domain is called the head and others
are called the body and the platform (Brimacombe et al.
1983; Wimberly et al. 2000). The head, body, and platform
domains of the SSU have direct functional significance,
moving independently during translation (Noller 2005).
These 3D domains may also have evolutionary significance.
The domain is the evolutionary unit of protein evolution
(Campbell and Downing 1994; Fong et al. 2007). Protein
domains are modular units that are combined and recom-
bined over evolution to achieve various functions. It is con-
ceivable that the 3D domains of the SSU played analogous
evolutionary roles, but on a more ancient timeframe. If so,
then the 3D domains of the SSU may have been recruited
to the ribosome, from prior functional roles.
The LSU in bacteria and archaea is made up of a 23S
rRNA and a much smaller 5S rRNA. The 23S rRNA con-
3
Corresponding author.
E-mail loren.williams@chemistry.gatech.edu.
Article published online ahead of print. Article and publication date are
at http://www.rnajournal.org/cgi/doi/10.1261/rna.030692.111.
752 RNA (2012), 18:752–758. Published by Cold Spring Harbor Laboratory Press. Copyright Ó2012 RNA Society.
tains six 2°domains (Fig. 1A; Noller et al. 1981). Although
these 2°domains are well-defined in the secondary structure,
in three dimensions the LSU appears monolithic (Tumminia
et al. 1994; Ban et al. 2000; Yusupov et al. 2001). It has been
suggested that, unlike in the SSU, the 2°domains in the LSU
do not correspond to 3D domains.
Questions naturally arise as to whether the architectures
and early evolution of the SSU and the LSU are fundamen-
tally different, and if so, why? Do isolated 2°domains of the
16S rRNA but not the 23S rRNA act as 3D domains and
fold to near-native 3D structures? How are the 2°domains
of the 16S and 23S rRNAs related to 3D structure, function,
and evolution of the ribosome?
Here we experimentally probe the domain structure of
the LSU. We show that one isolated 2°domain of the 23S
rRNA can fold to a near-native state in absence of the re-
FIGURE 1. (A) Secondary structure of the 23S rRNA of the large subunit of T. thermophilus (adapted with permission from Harry Noller). The
six secondary structural domains of 23S rRNA are shown: Domain I in gray, Domain II in brown, Domain III in pink, Domain IV in yellow,
Domain V in purple, and Domain VI in orange. (B) Tertiary interactions (dark blue) and phosphate–magnesium–phosphate linkages within
Domain III. Each first shell magnesium–phosphate interaction is indicated by a magenta circle. The lines between the circles are the phosphate–
magnesium–phosphate linkages. (C) SHAPE reactivities for Domain III
alone
in 250 mM Na
+
. The blue nucleotides are unreactive. (D)
Magnesium-dependent SHAPE reactivities for Domain III
alone
, observed upon addition of 10 mM Mg
2+
. Only the nucleotides with the greatest
proportional change in reactivity are indicated.
Domain III of the 23S rRNA is an independent fold
www.rnajournal.org 753
mainder of the LSU, and appears to be a true 3D domain.
Our focus here is Domain III of the Thermus thermophilus
23S rRNA (Fig. 1B), which is described by Thirumalai and
colleagues (Hyeon et al. 2006) as compact and slightly
prolate. We use SHAPE (Merino et al. 2005; Wilkinson
et al. 2005) to demonstrate that Domain III excised from
the 23S rRNA (Domain III
alone
) folds in a magnesium-
dependent fashion to the same basic state as when it is
embedded in the intact 23S rRNA (Domain III
23S
). In this
near-native state of Domain III, surface residues appear to
be poised with the correct geometry for the inter-domain
rRNA–rRNA interactions observed in the structure of the
LSU (PDB entry 2J01) (Selmer et al. 2006). Our results are
consistent with the structure of Domain III within the LSU
where Domain III is compact, and its interactions with
other ribosomal components are restricted to its surface (Figs.
2, 3; Supplemental Fig. S1).
RESULTS
SHAPE accurately predicts the canonical secondary
structure of Domain III
alone
The canonical secondary structure of the 23S rRNA, based
on comparative sequence analysis (Yusupov et al. 2001;
Cannone et al. 2002), is strongly supported by previous
SHAPE experiments (Deigan et al. 2009). As shown by
Weeks and colleagues, SHAPE exploits the reactivity of the
29-hydroxyl groups of RNA with electrophilic chemical
probing reagents such as NMIA (N-methylisatoic anhydride)
or BzCN (benzoyl cyanide) (Merino et al. 2005; Wilkinson
et al. 2005). The relative reactivities of the 29-hydroxyl
groups of various nucleotides are sensitive primarily to
local RNA flexibility. Consequently, paired nucleotides within
helical regions are generally less flexible and less reactive to-
ward SHAPE reagents than unpaired nucleotides.
TheclosecorrespondenceofourSHAPEdatatothe
canonical secondary structure of Domain III is illustrated in
Figure 1C, where SHAPE reactivity of Domain III
alone
is
mapped onto the canonical secondary structure. All SHAPE
reactivity data were obtained using NMIA unless otherwise
specified. The definition of Domain III used here is con-
ventional and includes residues G1271–G1647 of the 23S
rRNA by the Escherichia coli numbering scheme (Brosius
et al. 1980; Yusupov et al. 2001). These data were obtained in
presence of 250 mM Na
+
ions and in the absence of divalent
cations. Under these conditions, RNA is expected to assume
secondary structure but not necessarily tertiary structure
(Brion and Westhof 1997; Draper 2008). Consistent with this
tendency, the correspondence between SHAPE reactivities
and the secondary structure is very nearly perfect. Nucleo-
tides of Domain III
alone
were ranked using their absolute
SHAPE reactivities relative to A1572 (highest reactivity) and
binned into four groups, which are indicated in Figure 1C
(see Supplemental Material for a more detailed analysis).
Folding of Domain III
alone
to a near-native state
requires magnesium ions
The folding of RNAs from secondary structure to their
native states, containing long-range tertiary interactions, is
known to be generally magnesium-dependent (Brion and
Westhof 1997; Draper 2008). The native state of Domain
III rRNA, as inferred from the 3D structure of the as-
FIGURE 2. Domain III is compact and is not penetrated by other 2°
domains. (A) All six 2°domains of the 23S rRNA are shown, colored
as in Figure 1. Three views, with a relative rotation of 90°, are shown.
(B–F) Interactions of Domain III with Domains I, II, IV, V, and VI,
respectively.
Athavale et al.
754 RNA, Vol. 18, No. 4
sembled LSU, is stabilized by extensive networks of intra-
domain tertiary base–base, base–backbone, and backbone–
magnesium–backbone interactions (Fig. 1B). Consistent
with this observation, Figure 1D shows that the magnesium-
induced changes in SHAPE reactivity of Domain III
alone
are
widely dispersed over Domain III rRNA. The SHAPE reac-
tivities increase at some sites and decrease at others. The
nucleotides with SHAPE reactivities that are most sensitive
to magnesium are mapped onto the secondary structure in
Figure 1D. This magnesium dependence of the SHAPE
reactivity reflects (i) specific magnesium binding, (ii) more
diffuse interactions of magnesium with the RNA, and (iii)
tertiary rRNA–rRNA intra-domain interactions (Supple-
mental Tables S1, S2). Such magnesium-dependent SHAPE
reactivity has previously been demonstrated for tRNA
and RNase P (Merino et al. 2005; Mortimer and Weeks
2008).
We used two chemical reagents to verify that the observed
changes in reactivity are the result of RNA folding and not
from direct modulation of the reagent activity by magne-
sium. Although SHAPE reactivity of NMIA has been shown
to be modestly sensitive to magnesium (Mortimer and Weeks
2007), reactivity of BzCN is independent of magnesium
(Mortimer and Weeks 2008). We confirmed that NMIA
and BzCN show similar changes in SHAPE reactivity
upon addition of magnesium (Supplemental Fig. S2).
The secondary structure of Domain III rRNA
is conserved upon excision from the 23S rRNA
Figure 4A shows the SHAPE reactivities of Domain III
alone
and Domain III
23S
, both in the absence of magnesium. As
illustrated by the overlaid traces, the reactivities are essentially
identical along the length of the Domain III sequence. The
high degree of similarity suggests that the secondary structure
of Domain III
alone
is the same as Domain III
23S
.
Mg
2+
-mediated folding of Domain III to the near-native
state is conserved upon excision from the 23S rRNA
In the presence of magnesium ions, the SHAPE reactivities
for Domain III
alone
and Domain III
23S
are very similar (Fig.
4B). The magnesium-dependent state of Domain III is
therefore retained when it is excised from the 23S rRNA.
The data also show that inter-domain rRNA–rRNA in-
teractions are disrupted upon excision of Domain III from
the 23S rRNA. In presence of magnesium, the SHAPE
reactivity of Domain III
23S
differs subtly from that of Do-
main III
alone
(Fig. 4B). The differences are statistically focused
at nucleotides involved in inter-domain interactions in the
LSU, rather than at other regions of the Domain III rRNA.
Of the 33 nucleotides (nt) that report a difference in SHAPE
reactivity of $40% between Domain III
23S
and Domain
III
alone
in the presence of magnesium, 25 are seen to be
involved in direct inter-domain interactions (<3.4 A
˚
FIGURE 3. Domain III is not penetrated by ribosomal proteins. (A)
Domain III, colored and oriented as in Figure 2, with rProteins L2 (dark
blue), L17 (light blue), L22 (dark green), L23 (yellow), L24 (light brown),
and L34 (light green). (B–G) Interactions of Domain III with each of
these rProteins.
Domain III of the 23S rRNA is an independent fold
www.rnajournal.org 755
interatomic distances) in the LSU or are in close proximity to
those nucleotides involved in inter-domain interactions.
This pattern suggests that Domain III
alone
folds into a near-
native state, and that ‘‘insertion’’ of Domain III into the
23S rRNA (to form Domain III
23S
) primarily affects the
nucleotides involved in inter-domain interactions. A de-
tailed list of other inter-domain interactions is available in
Supplemental Tables S3, S4; the tables also indicate if
SHAPE detects these interactions.
Specifically, the 3D structure of the LSU shows that
A1284 forms base–backbone hydrogen bonds with G489 of
Domain I. Nearby A1287 forms base–base stacking inter-
actions with C1648 of Domain IV. As seen in Figure 4B,
adding Domain III back into the 23S rRNA changes the
SHAPE reactivities of A1284 and A1287. Similar changes in
SHAPE reactivities are seen for (i) fragment G1325–G1332,
where G1325 forms base–backbone hydrogen bonds with
A1269 and C1270 of Domain II, and U1326 forms base–
backbone hydrogen bonds with C1648 and G2010 of Do-
main IV; (ii) A1365, which forms base–backbone hydrogen
bonds with G187 of Domain I; (iii) nucleotides G1568–
A1570, where A1569 forms van der Waals contacts with
C693 of Domain II; and (iv) nucleotides A1616–C1617,
where C1617 forms base–backbone and
backbone–backbone hydrogen bonds
with C749 and A750 of Domain II. This
pattern of differential SHAPE reactivity
indicates the subtle structural changes
that occur when Domain III forms inter-
domain interactions with other elements
of the 23S rRNA. These inter-domain
interactions are disrupted when Domain
III is excised from the 23S rRNA, while
the intra-domain interactions are con-
served.
DISCUSSION
The domain structures of rRNAs have
profound implications for folding and
function of the ribosome, and early evo-
lution of life. In contrast to the SSU, it
has been proposed that the 2°domains
of the LSU (Fig. 1A) are melded into a
single monolithic unit (Tumminia et al.
1994; Ban et al. 2000; Yusupov et al.
2001). LSU 2°domains are thought to
be so highly intertwined and intercon-
nected that they lack distinct structural
and functional significance and are not
true 3D domains.
Considering the extensive network of
intra-domain tertiary interactions of Do-
main III (Fig. 1B; Supplemental Tables
S1, S2) and its isolation from the inter-
domain network of molecular interactions within the LSU,
we hypothesize that Domain III is a true 3D domain. In
contrast, Domain V, which contains the Peptidyl Trans-
ferase Center, is extensively networked with other 2°domains.
Domain V makes 24 inter-domain A-minor interactions
(Bokov and Steinberg 2009). Additionally, Domain V makes
six inter-domain magnesium-mediated phosphate–phos-
phate linkages (Hsiao and Williams 2009). Domain III
only makes six A-minor interactions and single magne-
sium-mediated phosphate–phosphate linkage with other 2°
domains.
We present data indicating that Domain III
alone
adopts
a secondary structure that is the same as Domain III
23S
(Figs. 1C, 4A). The addition of magnesium facilitates
folding to a near-native state of both Domain III
alone
and
Domain III
23S
, with the formation of intra-domain tertiary
interactions (Figs. 1D, 4B). The disruption of inter-domain
interactions of Domain III is reflected in the subtle but
observable changes in SHAPE reactivity when Domain III is
excised from the 23S rRNA (i.e., when Domain III
23S
is
converted to Domain III
alone
) (Fig. 4B). The mapping of
these changes in SHAPE reactivity to regions of inter-domain
interactions is evidence that Domain III
alone
and Domain
FIGURE 4. SHAPE reactivity for Domain III
alone
(blue) and Domain III
23S
(red). The vertical
axis represents SHAPE reactivities and the horizontal axis represents nucleotide position using
conventional E. coli numbering scheme. (A) Domain III
alone
and Domain III
23S
in 250 mM
Na
+
.(B) Domain III
alone
and Domain III
23S
in 250 mM Na
+
and 10 mM Mg
2+.
The inter-
domain interactions between Domain III and Domains I, II, and IV that cause differences in
SHAPE reactivity between Domain III
alone
and Domain III
23S
are highlighted. Hydrogen bonds
are shown by dashed lines, stacking interactions are shown by hashing, and van der Waals
contacts are shown by broad shaded arrows.
Athavale et al.
756 RNA, Vol. 18, No. 4
III
23S
fold to near-native states. This interpretation is sup-
ported by the previous observation that Domain III
alone
interacts specifically with ribosomal protein L23 (Ostergaard
et al. 1998).
In sum it appears that, like the SSU, the LSU also con-
tains some elements of a 3D domain-based architecture, in
spite of its monolithic appearance. At least some 2°domains
of the 23S rRNA (Domain III) autonomously fold to near-
native states apart from the rest of the LSU. Consequently,
at least some LSU 2°domains may have played roles similar
to SSU 2°domains during the evolutionary development of
the ribosome. Previous support for the importance of 3D
domains of the LSU is found in the demonstration that
Domain I alone is highly structured (Egebjerg et al. 1987).
Further, Garret and colleagues have demonstrated that
isolated domains of the 23S rRNA are able to form the
correct secondary structure and bind to specific ribosomal
proteins (Egebjerg et al. 1987; Leffers et al. 1988; Ostergaard
et al. 1998).
Evolutionary implications of the domain structure
of the Domain III
The ribosome in its present form was well-established at the
emergence of the last universal common ancestor of life
(LUCA) (Woese 2001; Wolf and Koonin 2007; Smith et al.
2008; Bokov and Steinberg 2009; Hsiao et al. 2009; Belousoff
et al. 2010; Fox 2010). There is a consensus that some parts
of the ribosome are even older than LUCA, predating the
protein world. Parts of Domain V of the 23S rRNA are
believed to be among the most ancient parts of the ribosome
(Woese 2001; Wolf and Koonin 2007; Smith et al. 2008;
Bokov and Steinberg 2009; Hsiao et al. 2009; Belousoff et al.
2010; Fox 2010) while Domain III is thought to be a more
recent addition (Hury et al. 2006). The data presented here
support the hypothesis that Domain III was added as an
intact entity to the ancestral ribosome—assuming that the 3D
domain is a unit of ribosomal evolution. This evolutionary
model is consistent with the absence of Domain III from
certain mitochondrial rRNAs, such as that of Trypanosoma
brucei (Sloof et al. 1985). Ribosomes in which Domain III is
absent may have had this domain deleted by relatively
recent evolutionary processes within the mitochondrion,
but presumably retained functionality with the assistance of
proteins.
MATERIALS AND METHODS
T. thermophilus rRNA transcripts were produced and purified as
described in the Supplemental Material.
SHAPE reactions
Magnesium was removed from 25 pmol of Domain III or 23S
RNA in 32 mL13TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) by
heating in the presence of magnesium chelating resin (Hampton
Research) to 95°C for 3 min, followed by chilling on ice. Thirty-
two microliters of Mg
2+
-free RNA was mixed with 4 mL103
folding buffer (500 mM HEPES pH 8.0, 2 M sodium acetate pH
8.0) and then incubated at 37°C for 20 min. For RNA folding with
Mg
2+
, the 103folding buffer contained 500 mM HEPES pH 8.0,
2 M sodium acetate pH 8.0, 100 mM MgCl
2
.
The folded RNA was divided equally between two tubes. To one
tube, 2 mL of 130 mM NMIA (or 800 mM BzCN) in anhydrous
DMSO was added, while the other half served as a negative control
to which 2 mL pure DMSO was added. The reactions were in-
cubated at 37°C for 1 h with NMIA. The modification reaction
using BzCN is complete in a few seconds at room temperature
(Mortimer and Weeks 2008). Denaturing SHAPE experiments
were performed in 20 mM HEPES pH 8.0 (final concentration)
for 4 min at 90°C using 130 mM NMIA in anhydrous DMSO. The
modified RNA was purified using RNeasy Mini Kit (Qiagen) and
resuspended in 20 mL13TE. The recovery after purification was
65%–75%.
A 20-nt long DNA oligomer 59-CGCGCCTGAGTGCTCTT
GCA-39, that anneals to the 39-end of Domain III, was used to
prime the reverse transcription. The primer was labeled with 6-FAM
using a 59-amino C6 linker (Operon MWG). Twenty microliters
of modified RNA was added to 8 pmol of the primer in 10 mLof
13TE. The RNA-primer solution was heated to 95°C for 1 min
and cooled to 30°C over 45 min at a rate of 1.4°C/min. After
primer annealing, SuperScript III Reverse Transcriptase buffer
(Invitrogen) was added at 30°C. The solution was heated to 55°C
for 1 min and reverse transcription was initiated by adding 1 mL
(200 U) of SuperScript III Reverse Transcriptase (Invitrogen). The
reaction was incubated at 55°C for 2 h and quenched by heating
to 70°C for 15 min. Di-deoxy sequencing reactions used
unmodified Domain III RNA and 1 mM ddNTPs (TriLink
BioTechnologies). One microliter of the reverse transcription re-
action mixture was mixed with 0.3 mL ROX-labeled DNA sizing
ladder and 8.7 mL of Hi-Di Formamide (Applied Biosystems) in a
96-well plate. The mixture was heated to 95°C for 5 min to de-
nature the cDNAs and resolved on a 3130 Genetic Analyzer
(Applied Biosystems) using custom fluorescence spectral calibra-
tion. Capillary electrophoresis data were processed as described in
the Supplemental Material.
Tertiary interactions
A detailed description of the protocol followed to annotate the
intra-domain and inter-domain tertiary interactions observed for
Domain III is available in the Supplemental Material.
SUPPLEMENTAL MATERIAL
Supplemental material is available for this article.
ACKNOWLEDGMENTS
This work was supported by the NASA Astrobiology Institute and
the Center for Ribosomal Origins and Evolution. We thank Timothy
K. Lenz for helpful discussions.
Received October 2, 2011; accepted December 24, 2011.
Domain III of the 23S rRNA is an independent fold
www.rnajournal.org 757
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