Effects of Noncanonical Base Pairing on RNA Folding: Structural Context and Spatial Arrangements of G·A Pairs

Abstract

Noncanonical base pairs play important roles in assembling the three-dimensional structures critical to the diverse functions of RNA. These associations contribute to the looped segments that intersperse the canonical double-helical elements within folded, globular RNA molecules. They stitch together various structural elements, serve as recognition elements for other molecules, and act as sites of intrinsic stiffness or deformability. This work takes advantage of new software (DSSR) designed to streamline the analysis and annotation of RNA three-dimensional structures. The multiscale structural information gathered for individual molecules, combined with the growing number of unique, well-resolved RNA structures, makes it possible to examine the collective features deeply and to uncover previously unrecognized patterns of chain organization. Here we focus on a subset of noncanonical base pairs involving guanine and adenine and the links between their modes of association, secondary structural context, and contributions to tertiary folding. The rigorous descriptions of base-pair geometry that we employ facilitate characterization of recurrent geometric motifs and the structural settings in which these arrangements occur. Moreover, the numerical parameters hint at the natural motions of the interacting bases and the pathways likely to connect different spatial forms. We draw attention to higher-order multiplexes involving two or more G·A pairs and the roles these associations appear to play in bridging different secondary structural units. The collective data reveal pairing propensities in base organization, secondary structural context, and deformability and serve as a starting point for further multiscale investigations and/or simulations of RNA folding.

Figure 1.

Comparison of the hydrogen-bonding interactions, chemical structures, and relative spatial arrangements of nucleotides in the six dominant modes of G·A pairing and in a canonical Watson-Crick G−C pair. Hydrogen bonds shown by thin dashed lines, with arrows directed toward base/backbone atoms capable of accepting protons. Structures generated with 3DNA and rendered in PyMOL (www.pymol.org) using the average rigid-body parameters in Table 1 and a canonical A-RNA backbone. Structures depicted in the standard reference frame of G. Color-coding denotes the mode of base association: sheared m−M (dark blue); imino W−W (gray); m+m (pink); m−m (red); m+W (light blue); m−W (magenta); canonical (white), where the combinations of signs and letters denote the orientation (parallel +/antiparallel −) and the approximate sites (minor m/major M/Watson-Crick W edges) of base association. Interestingly, less than 10% of the + states reflect an anti-to-syn sugar-base rearrangement, and these examples all involve adenine.

Figure 2.

Molecular images of RNA secondary structural motifs incorporating each of the dominant modes of G·A base pairing. Motifs correspond to one of the common settings of the designated pairs: (a) sheared m−M pair closing the GNRA tetraloop at the end of the P4 helix of the glmS ribozyme bound to glucosamine 6-phosphate; (b) imino W−W pair at the end of an asymmetric internal loop in the Thermus thermophilus 70S ribosome in complex with the hibernation factor pY; (c) m+m pair linking the G in a double-helical stem and the A in an internal loop of the complex of Thermus thermophilus ribosomal protein L1 with a fragment of the L1 RNA from Methanoccocus vannielii; (d) m−m pair joining a stem and single-stranded fragment within a three-way junction of the Thermoanaerobacter tengcongenesis ydaO riboswitch bound to cyclic di-AMP; (e) m+W pair connecting the D and T hairpin loops of yeast initiator transfer RNA; (f) m−W pair linking two hairpin loops of the 5S rRNA within the structure of the hibernating 100S ribosome dimer from pathogenic Staphylococcus aureus. G·A pairs color-coded as in Figure 1 and shown at the nucleotide level within each structural element and in a separate local depiction to the right of each example. Loops depicted in gold and canonical base pairs at the ends of loops or within double-helical stems in white. Ribbons connect phosphorus atoms in successive nucleotides. See Table S3 for respective Protein Data Bank identifiers, chain names, and residue numbers of the depicted pairs and Figure S2 for simple secondary structural diagrams of the associated motifs.

Figure 3.

Scatter plots of the rigid-body components — Shear, Stretch, Opening — that distinguish the modes of G·A association in RNA-containing structures. Smooth curves on the edges of the scatter plots are the normalized densities of individual parameters. Points with the magnitude of Opening in excess of 180° include requisite changes in the signs of Shear, Stretch, Buckle, and Propeller. Color-coding of dominant pairs matches that in Figures 1, 2. Secondary states with 16 or more structural examples are noted by related hues. Images depict the spread of values in the Shear-Opening (left) and Shear-Stretch (right) planes for antiparallel G−A (top) and parallel G+A (bottom) arrangements.

Figure 4.

Succession of configurations illustrating the rigid-body motions that transform the associations of G and A between different pairing modes. Images of adenine oriented with respect to a common coordinate frame on guanine. Structures generated with 3DNA using the average rigid-body parameters reported in Table 1. Base pairs color-coded by interaction mode (Figures 1-3), with the minor (II) substates of m±W and m−M pairs noted by lighter hues. Pathways connecting (a) antiparallel m−m, m−W_I, m−W_II, W−W, m−M_I, m−M_II states; (b) parallel m+m, m+W_II, m+W_I, m+M states. Note the counterclockwise rotation of ribose C1′ atoms (darkened spheres) along the top-to-bottom transformation of antiparallel pairs and the clockwise rotation along the corresponding progression of parallel pairs. Hydrogen bonds between base atoms depicted by thin dashed lines.

Figure 5.

Molecular images illustrating hydrogen bonds (dashed lines) shared between different forms of G·A pairing: (a) the N3⋯N6 interaction common to m−W_II and m−M_I base-paired arrangements found respectively in a variant of the SAM-I riboswitch and the complex of Escherichia coli ribosomal protein L25 with a 5S rRNA fragment; (b) the respective N2⋯N1 associations in m−W_I and W−W pairs in the Leishmania donovani large ribosomal subunit and the Saccharomyces cerevisiae 80S ribosome; (c) the N2⋯N7, N1⋯OP2, and N2⋯OP2 hydrogen bonds respectively stabilizing W−M and m−M_II pairs in the central domain of the Thermus thermophilus 30S ribosomal subunit and in the complex of the Thermus thermophilus 70S ribosome with hibernation factor pY; (d) the respective paired association of 2′-hydroxyl groups, one from G and the other from A, adopted in m−m and m−W_I pairs within the complex of tetracycline with the U1052G-mutated 70S Escherichia coli ribosome.

Figure 6.

Molecular images of multiplets with two or more modes of G·A pairing in the complex of tetracycline with the U1052G-mutated 70S Escherichia coli ribosome. Loops incorporating m−M sheared pairs are linked by different associations of G and A (highlighted within boxes) to other secondary structural units. A local representation of the linked bases is shown below each global depiction of associated secondary structures. Examples include: (a) a tetraplex with m+m pairing between a hairpin loop and a double-helical stem; (b) a tetraplex with m+m pairing between an internal loop and a double-helical stem; (c) a pentaplex with m+W pairing between a hairpin loop and a double-helical stem that is linked in turn to a junction; (d) a tetraplex with m−m link pairing between an internal loop and a 5-way junction; (e) a triplex with m−W pairing between a hairpin loop and a 3-way junction; (f) a pentaplex with .–M and m−W pairing within a 7-way junction. G·A pairs and secondary structural motifs color-coded as in Figure 2. See Table S3 for respective Protein Data Bank identifiers, chain names, and residue numbers of depicted G·A-linked multiplets. Color-coding of bases and hydrogen bonds matches that in the corresponding secondary structural diagrams in Figure S8.

Figure 7.

Histograms of G·A deformability expressed in terms of the volume of conformation space occupied by the two bases in the major pairing schemes: (a) comparative contributions of pattern-specific (Shear, Stretch, Opening – red bars), out-of-plane (Stagger, Buckle, Propeller – green bars), and all six rigid-body parameters (blue bars – values listed in Table 1) to base-pair mobility; (b) relative deformability of G·A pairs in different tertiary structural settings, each with 44 or more structural examples. Base pairs grouped in terms of the observed setting of G followed by that of A. Note the logarithmic scale of volume and the tenfold (×10) enhancement of pattern-specific values.