Recurrent structural RNA motifs, Isostericity Matrices and sequence alignments

Abstract

The occurrences of two recurrent motifs in ribosomal RNA sequences, the Kink-turn and the C-loop, are examined in crystal structures and systematically compared with sequence alignments of rRNAs from the three kingdoms of life in order to identify the range of the structural and sequence variations. Isostericity Matrices are used to analyze structurally the sequence variations of the characteristic non-Watson-Crick base pairs for each motif. We show that Isostericity Matrices for non-Watson-Crick base pairs provide important tools for deriving the sequence signatures of recurrent motifs, for scoring and refining sequence alignments, and for determining whether motifs are conserved throughout evolution. The systematic use of Isostericity Matrices identifies the positions of the insertion or deletion of one or more nucleotides relative to the structurally characterized examples of motifs and, most importantly, specifies whether these changes result in new motifs. Thus, comparative analysis coupled with Isostericity Matrices allows one to produce and refine structural sequence alignments. The analysis, based on both sequence and structure, permits therefore the evaluation of the conservation of motifs across phylogeny and the derivation of rules of equivalence between structural motifs. The conservations observed in Isostericity Matrices form a predictive basis for identifying motifs in sequences.

Figure 1

Flow chart showing iterative process relating structure-based RNA motifs and accuracy of sequence alignments.

Figure 2

Stereographic view of a crystallographic structure of a typical Kink-turn (14,28) with its annotated secondary structure following the nomenclature for non-WC pairs (3). Each characteristic base pair is circled in the 3D diagram with colors corresponding to those in the 2D diagram. The same color code is used to frame the Isostericity Matrix attached to each base pair. Base pair 1 (BP1), colored orange, is cis-WC/WC; Base pair 2 (BP2), in red, is trans-H/SE; Base pair 3 (BP3), in purple, is trans-H/SE; Base pair 4 (BP4), in blue, is trans-SE/SE; Base pair 5, in green, is trans-SE/SE. In each Isostericity Matrix, the families of isosteric pairs (I1, I2, etc.) have an identical colored background. Parentheses indicate modeled interactions for the isosteric relationships not yet observed in high-resolution X-ray structures (4).

Figure 3

Annotated secondary structures of Kink-turn motifs from crystal structures, comparing structural variants to the typical Kink-turn, exemplified by KT-7 from archaeal 23S rRNA. Each characteristic base pair is framed in a different color: The last base pair of the C-stem in orange (base pair 1), the two trans-H/SE base pairs of the NC-stem, base pair 2 in red and base pair 3 in purple, and the two trans-SE/SE, base pair 4 in blue and base pair 5 in green. Each tertiary interaction is represented by a unique symbol indicating the interacting edges of the bases and whether the pair is cis or trans (3).

Figure 4

Upper panel: Annotated secondary structures of the conserved 23S rRNA Kt-46 Kink-turn motif. Lower panel: Isostericity Matrix analysis of characteristic base pairs of Kt-46. In each box, the percentage of observed sequences for each base pair is given for Archaea (A), Bacteria (B) and Eukarya (E) (upper, middle and lower).

Figure 5

Aggregated tables of sequence variations for each of the characteristic base pairs of conserved Kink-turns. The data for the conserved Kink-turns are given. These serve to define the sequence signatures for Kink-turn motifs. For pair 1 (orange cis-WC/WC), the number of sequences were A = 264, B = 5625, E = 10 846. For Pair 2 (red trans-H/SE), A = 240, B = 4820, E = 10 713; KT-77/78 was excluded, since pair 2 is absent. For Pair 3 (purple trans-H/SE), A = 168, B = 2415 and E = 266. The following K-turns were excluded: KT-4/5 where trans-H/SE is replaced by trans-H/H base pair; KT-42 where trans-H/SE is absent in X-ray structure although bases are present; KT-23 where trans-H/SE is replaced by trans-WC/H base pair (see below); KT-11 where trans-H/SE is replaced by trans-WC/H base pair (see below). For Pair 3 (purple trans-WC/H), A = 48, B = 1600 and E = 10 377. KT-11 and KT-23 were taken into account. For Pair 4 (blue trans-SE/SE), A = 216, B = 5625 and E = 10 846. The following were excluded: KT-58 where pair 4 is absent in the structure; KT-15 where trans-SE/SE is replaced by a cis-SE/SE base pair. For Pair 5 (green trans-SE/SE), A = 264, B = 5625 and E = 10 846.

Figure 6

Stereographic view of a crystallographic structure of a typical C-loop (14,28) with its annotated secondary structure following the nomenclature for non-WC pairs (3). Each characteristic base pair is circled in the 3D diagram with colors corresponding to those in the 2D diagram. The same color code is used to frame the Isostericity Matrix attached to each base pair. Base pair 1 (BP1) is colored red: cis-WC/WC; Base pair 2 (BP2) in purple: trans-WC/H; Base pair 3 (BP3) in blue: cis-WC/SE; Base pair 4 (BP4) in green: cis-WC/WC. In each Isostericity Matrix, the families of isosteric pairs (I1, I2, etc.) have an identical colored background. Parentheses indicate modeled interactions for the isosteric relationships not yet observed in high-resolution X-ray structures (4).

Figure 7

Annotated secondary structures for C-loop motifs from crystal structures, comparing structural variants with a typical C-loop, exemplified by C15 from helix 15 of 16S rRNA. The four characteristic interactions of this motif are shown: canonical basepairs 1 in red and 4 in green, trans-WC/H base pair 2 in purple and cis-WC/SE base pair 3 in blue.

Figure 8

Upper panel: Annotated secondary structures of 16S C96, for representative archaeal, bacterial and eukaryal sequences. Lower panel: Isostericity Matrix analysis of characteristic base pairs of 16S C96. In each box, the percentage of observed sequences for each base pair is given for Archaea, Bacteriae and Eukarya (upper, middle and lower).

Figure 9

Aggregated tables of sequence variations for each of the characteristic base pairs of conserved C-loops.

Figure 10

Covariation tables for two additional pairs present in the C-loop motifs 23S C38 and 16S C15.

Figure 11

Annotated secondary structures for two C-like motifs, the C-like 28 from the 16S rRNA and loop C in the 5S rRNA. Although the overall 3D fold is maintained, the distinctive C of the trans-WC/H pair is absent in those motifs. An additional pair is present (black). In grey are shown the usual pairs of the C-motifs.