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. 2012 Apr;18(4):752–758. doi: 10.1261/rna.030692.111

Domain III of the T. 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, Loren Dean Williams 2,3
PMCID: PMC3312562  PMID: 22334759

The ribosomal 23S rRNA is contained in six secondary structure domains. The authors show that Domain III is a truly independent structural domain of the large subunit (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. The SHAPE data presented support previous suggestions that Domain III was added relatively late in ribosomal evolution.

Keywords: ribosome, large subunit, independent fold, ribosomal evolution, SHAPE

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 2′-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.

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 contains 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 independent and autonomous three-dimensional domains (3D domains) 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° domains (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 recombined over evolution to achieve various functions. It is conceivable 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 contains 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.

FIGURE 1.

FIGURE 1.

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(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 IIIalone in 250 mM Na+. The blue nucleotides are unreactive. (D) Magnesium-dependent SHAPE reactivities for Domain IIIalone, observed upon addition of 10 mM Mg2+. Only the nucleotides with the greatest proportional change in reactivity are indicated.

Questions naturally arise as to whether the architectures and early evolution of the SSU and the LSU are fundamentally 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 remainder 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 IIIalone) folds in a magnesium-dependent fashion to the same basic state as when it is embedded in the intact 23S rRNA (Domain III23S). 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).

FIGURE 2.

FIGURE 2.

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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.

FIGURE 3.

FIGURE 3.

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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.

RESULTS

SHAPE accurately predicts the canonical secondary structure of Domain IIIalone

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 2′-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 2′-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 toward SHAPE reagents than unpaired nucleotides.

The close correspondence of our SHAPE data to the canonical secondary structure of Domain III is illustrated in Figure 1C, where SHAPE reactivity of Domain IIIalone 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 conventional 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. Nucleotides of Domain IIIalone 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 IIIalone 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 assembled 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 IIIalone are widely dispersed over Domain III rRNA. The SHAPE reactivities 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 (Supplemental 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 magnesium. 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 IIIalone and Domain III23S, 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 IIIalone is the same as Domain III23S.

FIGURE 4.

FIGURE 4.

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SHAPE reactivity for Domain IIIalone (blue) and Domain III23S (red). The vertical axis represents SHAPE reactivities and the horizontal axis represents nucleotide position using conventional E. coli numbering scheme. (A) Domain IIIalone and Domain III23S in 250 mM Na+. (B) Domain IIIalone and Domain III23S in 250 mM Na+ and 10 mM Mg2+. The inter-domain interactions between Domain III and Domains I, II, and IV that cause differences in SHAPE reactivity between Domain IIIalone and Domain III23S 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.

Mg2+-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 IIIalone and Domain III23S 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 interactions are disrupted upon excision of Domain III from the 23S rRNA. In presence of magnesium, the SHAPE reactivity of Domain III23S differs subtly from that of Domain IIIalone (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 III23S and Domain IIIalone in the presence of magnesium, 25 are seen to be involved in direct inter-domain interactions (<3.4 Å interatomic distances) in the LSU or are in close proximity to those nucleotides involved in inter-domain interactions. This pattern suggests that Domain IIIalone folds into a near-native state, and that “insertion” of Domain III into the 23S rRNA (to form Domain III23S) primarily affects the nucleotides involved in inter-domain interactions. A detailed 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 interactions 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 Domain 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 conserved.

DISCUSSION

The domain structures of rRNAs have profound implications for folding and function of the ribosome, and early evolution 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 interconnected that they lack distinct structural and functional significance and are not true 3D domains.

Considering the extensive network of intra-domain tertiary interactions of Domain 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 Transferase 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–phosphate linkages (Hsiao and Williams 2009). Domain III only makes six A-minor interactions and single magnesium-mediated phosphate–phosphate linkage with other 2° domains.

We present data indicating that Domain IIIalone adopts a secondary structure that is the same as Domain III23S (Figs. 1C, 4A). The addition of magnesium facilitates folding to a near-native state of both Domain IIIalone and Domain III23S, 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 III23S is converted to Domain IIIalone) (Fig. 4B). The mapping of these changes in SHAPE reactivity to regions of inter-domain interactions is evidence that Domain IIIalone and Domain III23S fold to near-native states. This interpretation is supported by the previous observation that Domain IIIalone interacts specifically with ribosomal protein L23 (Ostergaard et al. 1998).

In sum it appears that, like the SSU, the LSU also contains 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 μL 1× TE (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 Mg2+-free RNA was mixed with 4 μL 10× 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 Mg2+, the 10× folding buffer contained 500 mM HEPES pH 8.0, 2 M sodium acetate pH 8.0, 100 mM MgCl2.

The folded RNA was divided equally between two tubes. To one tube, 2 μL 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 μL pure DMSO was added. The reactions were incubated 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 μL 1× TE. The recovery after purification was 65%–75%.

A 20-nt long DNA oligomer 5′-CGCGCCTGAGTGCTCTTGCA-3′, that anneals to the 3′-end of Domain III, was used to prime the reverse transcription. The primer was labeled with 6-FAM using a 5′-amino C6 linker (Operon MWG). Twenty microliters of modified RNA was added to 8 pmol of the primer in 10 μL of 1× TE. 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 μL (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 reaction mixture was mixed with 0.3 μL ROX-labeled DNA sizing ladder and 8.7 μL of Hi-Di Formamide (Applied Biosystems) in a 96-well plate. The mixture was heated to 95°C for 5 min to denature the cDNAs and resolved on a 3130 Genetic Analyzer (Applied Biosystems) using custom fluorescence spectral calibration. 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.

Footnotes

Article published online ahead of print. Article and publication date are at http://www.rnajournal.org/cgi/doi/10.1261/rna.030692.111.

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