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The GA‐minor submotif as a case study of RNA modularity, prediction, and design

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Abstract Complex natural RNAs such as the ribosome, group I and group II introns, and RNase P exemplify the fact that three‐dimensional (3D) RNA structures are highly modular and hierarchical in nature. Tertiary RNA folding typically takes advantage of a rather limited set of recurrent structural motifs that are responsible for controlling bends or stacks between adjacent helices. Herein, the GA minor and related structural motifs are presented as a case study to highlight several structural and folding principles, to gain further insight into the structural evolution of naturally occurring RNAs, as well as to assist the rational design of artificial RNAs. WIREs RNA 2013, 4:181–203. doi: 10.1002/wrna.1153 This article is categorized under: RNA Structure and Dynamics > RNA Structure, Dynamics, and Chemistry RNA Evolution and Genomics > RNA and Ribonucleoprotein Evolution

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GA‐minor submotif as building block for larger recurrent RNA motifs. Two‐dimensional sequence signatures, topological cartoons, and three‐dimensional stereo views of (a) RA motif (or RA turn), (b) kink‐turns, and (c) GA‐minor 2h_stack all containing the GA‐minor type I‐Tw submotif (highlighted in blue). The most significant structural characteristics of the motifs are indicated on their respective sequence signatures according to the annotation of Leontis and coworkers.6,77 For bps symbols: Watson–Crick, Hoogsteen, and shallow groove edges are indicated by circle, square, and triangle, respectively. For example, the HG:SG trans bp is symbolized by an open square associated to an open triangle. All SG:SG trans bp interactions between nts are represented by open triangles. Capital letters indicate that the nucleotide position is conserved in more than 90% of the cases; small letters indicate that the nucleotide position is conserved in more than 75% of the cases. Nn, a sequence of n nucleotides; 5′ and the arrow symbol indicate 5′ and 3′ ends, respectively. Note that the A‐minor 2h_stack is the former A‐minor triple motif mentioned in Ref 6.

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Definition and structural characteristics of the RA motif (or RA turn). (a) Nomenclature and generic sequence signature based on the structural analysis of RA motifs from known X‐ray structures.39 Tertiary interactions and noncanonical base pairs (bps) are indicated on the 2D diagram where open triangles represent SG:SG trans bp. SG:SG trans bps can sometimes be of type 1 twisted (indicated by T) or supertwisted (indicated by ST). SG:SG trans interactions in the along‐groove motif can be either symmetrical (indicated by =) or quasi‐symmetrical (indicated by ∼). Circled nts in red have C2′ endo sugar pucker. Capital letters indicate that the nucleotide position is conserved in more than 90% of the cases; small letters indicate that the nucleotide position is conserved in more than 75% of the cases. Open rectangles indicate stacking interactions; N, any nucleotide (A, U, C, or G); R or r, purine; Y or y, pyrimidine; 5′ and the arrow symbol indicate 5′ and 3′ ends, respectively. Additional nomenclature is defined in the legends of Figures 3 and 5. The regions colored in blue and rose highlight the ‘GA‐minor’ and ‘along‐groove’ components of the RA motif. The region in violet corresponds to the overlap of these two motifs. (b) Topological characteristics of the RA motif. The two adjacent helices H5′ and H3′ are oriented by 90° similarly to the corners of a log cabin. Nucleotide at position 13, at the 3′end of the motif, is in perfect helical continuity with H3′, allowing an additional helix to be stacked in continuity of this helix. (c) Three‐dimensional stereo image of the RA motif using the same color code as in (a) (from Ec_23S_RA.2). Note the quasi‐symmetrical arrangement of helices H5′ and H3′ and the perfect helical continuity existing between N13 and H3′. (d) Stereo image of the G:A SG:SG trans bps formed between A1 and R6 (type Stw) and between A7 and g12 (type Tw). Note the stacking interactions between the bases of A1, A7 and ribose of N13 (from Tt_23S_RA.1). (e and f) The along‐groove packing interactions with associated H‐bonds networks (from Hm_23S_RA.1): (e) symmetrical SG:SG bp contact between G2:C5 WC bp and G8:C11 WC bp (highlighted in yellow) and (f) quasi‐symmetrical SG:SG bp contact (highlighted in yellow) between Y3:R4 WC bp (G:U shown) and Y9:R10 WC bp. While R10 and R4 are often Gs, the position Y9 or Y4 can be either a U or a C. Typically, when Y3 is a C, Y4 is a U, or vice versa.

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The GA‐minor motif. (a) Comparison of A:G SG:SG trans bps of type I‐STw (magenta) and Tw (green) with the more classic A‐minor interaction of type I‐P planar (violet).37 (Left panel) Diagram that illustrates the two types of angles (tilt and propeller twist) that define the orientation of the planar, aromatic rings of two base‐paired nucleotides with respect to one another.74 (Right panel) The G positions of the A:G‐minor motifs listed in Table 2 were all superimposed with LSQMAN. The adenine position can adopt various propeller twist angles with respect to the G position. (b) 3D views of the four different categories of A‐minor interaction of type I: supertwisted (STw), twisted (Tw), planar (P), and tilted (T). The typical tilt and propeller twist angles are also indicated for each type of interaction (they can vary by ±15°). They all involve two H‐bonds. A‐minor type I‐P and type I‐T are most of the time associated with A‐minor type II interactions.37 The sugar pucker of the nucleotide in position A(1) is typically C3′ endo. By contrast, A‐minor type I‐Tw and type I‐STw are seldom or never associated with A‐minor type II interactions. The sugar pucker of the nucleotide position A(1) is typically C2′ endo. (c) 2D diagram and 3D view of the GA minor of type I‐STw: the G:A SG:SG trans bp of type I14,37 is supertwisted (STw) so that atom N1 of A interacts with the 2′ OH of G and atom N2 of G interacts with the 2′ OH of A. (d) 2D diagram and 3D view of the GA minor of type I‐Tw: the G:A SG:SG trans bp is twisted (Tw). Atom N3 of A interacts with atom N2 of G. Atom N1 of A interacts with 2′ OH of G. In addition, the adenine is stacked on the ribose of the nucleotide in 3′ of the G. While A(1) is highly conserved, the guanine position g(4) is less conserved and thus depicted as a lowercase ‘g’ on the 2D diagrams. Open triangles indicate SG:SG trans bps and open rectangles indicate stacking interactions. Circled nts in red have C2′ endo sugar pucker.

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Structural and functional equivalency of the RA‐2h_stack motif from the Tetrahymena group IC1 intron and the tail–tail interaction used in RNA tectosquares. The 2D (top) and 3D (bottom) diagrams of the RA‐2h_stack motif, previously reported in RNA tectosquares54 and later identified in the P2.1‐P3‐P8 junction of group IC1 and ID introns,39 demonstrate the inherent modularity associated with independently generated structures. On the left is shown a revised 3D model of the Tetrahymena group I intron39 with the P2.1‐P3‐P8 junction corresponding to the RA‐2h_stack (in blue). The directionality of the tectosquare assemblies is dictated by the pairing that takes place between the two exiting 3′ tails (seen on the right). The RA motif residing in individual tectosquare monomers assembling through their respective 3′ tails results in the formation of two RA‐2h_stack motifs. The GA‐minor motif present in the RA motif, in both cases, allows the 3′ tail to exit the RA in such a way that promotes the exiting strands ability to stack nascent helices.

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RA motifs and proteins. (a) In the 23S ribosomal RNA of E. coli (i), the interhelical G‐A connector of the H29‐H30 RA turn (magentas) is recognized by the ribosome‐binding protein L15 (green) (ii). (iii) Recognition by L15 involves the N‐H group from the peptide backbone belonging to Thr128, Lys129, and Gly30, the positively charged side chain of Lys109 as well as the negatively charged side chain of Glu76. (b) In the 23S ribosomal RNA of Haloarcula marismortui (i), the interhelical A‐A connector of the H29‐H30 RA turn (magentas) is recognized by the ribosome‐binding protein L15 (green), whereas the H27‐H28 RA turn (ruby) is recognized by the ribosome‐binding protein L18e (cyan) (ii). (iii) Recognition by L18e involves the N‐H group from peptide backbone belonging to Ser82, Gly83, and Thr84, the positively charged side chain of Lys63 and the negatively charged side chain of Glu42. The mode of recognition of these RA motifs by two different proteins L15 and L18e is strikingly similar.25 (c) (i) Superimposition of the C‐terminal domains of L15 and L18e. (ii and iii) Closer views. The striking similarity of the RA motif recognition domain of L15 and L18e suggests that they have a common evolutionary origin. This is corroborated by phylogenetic analysis.101

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The RA motif as signature for ribosomal evolution: a hypothetical scenario for the structural evolution of the H27‐H30 peripheral domain of the large rRNA of the large ribosomal subunit. (a) Before the major divergence of bacteria, archaea, and eukaryotes, local duplications of one of the GA‐minor elements H28 of an ancestral RA motif (H27‐H28) might have given rise to a four‐way junction domain formed by two RA motifs (H27‐H28 and H29‐H30). (b) While the ancestral four‐way RA junction is preserved in bacteria, it has undergone several additional structural changes in archaea, with the four‐way junction serving as scaffolding for further structural expansion. Insertion of additional sequences between positions 7 and 8 within the H29‐H30 RA in archaea might have transformed the four‐way junction into a four‐way junction. (c) Superposition of domains H27‐30 from bacteria [Ec_23S rRNA (PDB_ID: 2AW4)] and archaea [Hm_23S rRNA (PDB_ID: 1JJ2)].

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RNA modularity and the three principles of equivalence. (a) The principle of isosteric equivalence exemplified by the RA motif constructed from its elemental submotifs. Watson–Crick (WC) base pairing is the basis for isosteric equivalence. The closing base pair (bp) on the GA minor is superimposable to the WC bps of the along‐groove motif (overlaid with their electron densities in purple). The secondary structure and the stereo view of the RA motif are shown on the right. (b) The principle of structural equivalence as exemplified by the kink‐turn motif. The kink‐turn can take advantage of two different types of internal loops with different patterns of bps [GAA:gga (top) and loop E (bottom)]. The two types of kink‐turns (GA‐shared darker shade and loop E lighter) are shown overlaid in stereo (right). (c) The principle of topological/functional equivalence exemplified by three different RNA tectosquares. The individual motifs, the RA, the 3WJ, and the tRNA motif, while exhibiting completely different secondary structures and hydrogen‐bonding patterns provide similar 90° bends that form the corner of the tectosquares.

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The generation of motifs of increased complexity from the expansion of the GA minor. The RA turn, kink‐turn, and GA‐minor 2h‐stack motifs show their ability to accommodate additional motifs to increase structural complexity as exemplified in by the hydrogen‐bonding patterns found in several naturally occurring RNAs. While the kink‐turn can have insertions of additional sequence and helical elements within some of its modular components,72 kink‐turn motifs expanded through GA‐minor 2h_stack have not yet been identified. However, it is anticipated that a helix could be inserted within a kink‐turn and positioned in perfect helical continuity of H3′ like in the RA motif [see for example the remarkable structure of the kink‐turn in domain L1 of the Tt_23S rRNA (L1:H76‐78 from Tt_23S_rRNA [PDB_ID:1MZP]) that would allow this type of structural arrangement]. For bps and other symbols, see the legend in inset.

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The hierarchical syntax network of RNA structural motifs related to the GA‐minor submotif. Motifs are minimally sized recurrent set of nucleotides with conserved conformation. They are generally organized from the left to the right according to their increase in structural complexity. All motifs that comprise the GA‐minor motif as a building block are circled in blue. The most significant structural characteristics of the motifs are indicated on their respective sequence signatures according to the annotation of Leontis and coworkers.6,77 Visual of the three‐dimensional structures of some of these motifs can be found in the figures indicated in blue below the motif name. Points of connection to the previous ‘UA_h motif syntax network’6 are indicated by red stars. For bps and other symbols, see legend in inset: WC, Watson–Crick edge (circle); HG, Hoogsteen edge (square); SG, shallow groove edge (triangle). For example, the HG:SG trans bp is symbolized by an open square associated to an open triangle. The SG:SG cis bp is represented by a plain triangle. Nn, a sequence of n nucleotides; H and P stand for pairings; 5′ and the arrow symbol indicate 5′ and 3′ ends, respectively. See also legends of Figures 2 and 3. Note that the A‐minor 2h_stack is the former A‐minor triple motif mentioned in Ref 6.

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Variants of the combined GA‐minor and 2h_stack motifs: (a) local extension of the GA‐minor motif displayed in Figure 2 with GA‐minor bulge motifs. Three‐dimensional stereo views of (b) a GA‐minor bulge motif [A50‐G52‐U359‐A360 from Tt_16S rRNA (PDB_ID: 1J5E)] and (c) a GA‐minor bulge 2h_stack motif [A58‐C60‐G87‐G88 from GlmS ribozyme (PDB_ID: 2GCV)]. For annotation, see legends of Figures 2 and 3.

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