The application of cluster analysis in the intercomparison of loop structures in RNA

Abstract

We have developed a computational approach for the comparison and classification of RNA loop structures. Hairpin or interior loops identified in atomic resolution RNA structures were intercompared by conformational matching. The root-mean-square deviation (RMSD) values between all pairs of RNA fragments of interest, even if from different molecules, are calculated. Subsequently, cluster analysis is performed on the resulting matrix of RMSD distances using the unweighted pair group method with arithmetic mean (UPGMA). The cluster analysis objectively reveals groups of folds that resemble one another. To demonstrate the utility of the approach, a comprehensive analysis of all the terminal hairpin tetraloops that have been observed in 15 RNA structures that have been determined by X-ray crystallography was undertaken. The method found major clusters corresponding to the well-known GNRA and UNCG types. In addition, two tetraloops with the unusual primary sequence UMAC (M is A or C) were successfully assigned to the GNRA cluster. Larger loop structures were also examined and the clustering results confirmed the occurrence of variations of the GNRA and UNCG tetraloops in these loops and provided a systematic means for locating them. Nineteen examples of larger loops that closely resemble either the GNRA or UNCG tetraloop were found in the large ribosomal RNAs. When the clustering approach was extended to include all structures in the SCOR database, novel relationships were detected including one between the ANYA motif and a less common folding of the GAAA tetraloop sequence.

FIGURE 1.

UPGMA cluster analysis of the tetraloops (four base loop plus closing pair) found in 15 RNA molecules whose structure has been determined by X-ray crystallography. Individual loops are designated as described in the Materials and Methods. Three key branch points in the tree, which are discussed in the text, are labeled as A, B, and C. The distance scale at the bottom of the tree is in angstroms.

FIGURE 2.

(A) Dendrogram showing the similarity between GAAA and ANHA loops (H is A or C or U). Multiple examples of the same loop determined under slightly different experimental conditions (i.e., redundant loops) are included in this particular figure to illustrate how the methodology handles them. (B) Superimposition of a GAAA loop versus an AUUA loop. Black color for AUUA:1zdi:r:9–12 and gray color for GAAA:1nwx:0:122–125; closing base pairs are also shown.

FIGURE 2.

(A) Dendrogram showing the similarity between GAAA and ANHA loops (H is A or C or U). Multiple examples of the same loop determined under slightly different experimental conditions (i.e., redundant loops) are included in this particular figure to illustrate how the methodology handles them. (B) Superimposition of a GAAA loop versus an AUUA loop. Black color for AUUA:1zdi:r:9–12 and gray color for GAAA:1nwx:0:122–125; closing base pairs are also shown.

FIGURE 3.

UPGMA dendrogram showing clustered tetraloops and tetraloop-like longer hairpin loops. Individual loops are designated as described in the Materials and Methods. The distance scale at the bottom of the tree is in angstroms. GNRA tetraloops are highlighted in blue and the GNRA-like pentaloops are highlighted in yellow. UNCG tetraloops are highlighted in light green and the UNCG-like pentaloops are highlighted in dark green. Pentaloops occurring in nonribosomal RNAs that were previously identified as resembling either GNRA or UNCG tetraloops (Legault et al. 1998; Huppler et al. 2002; Theimer et al. 2003) are highlighted in purple. Hexaloops that closely resemble the GNRA tetraloop are highlighted in gray. Four tetraloop-like heptaloops are highlighted in orange. Pink-shaded loops resemble the ANYA tetraloop. To keep the total length for all the hairpin loops the same, the bulged out base in the 7-nt pentaloops, the closing pair of the 8-nt hexaloops, and both the bulged base and closing pair of the 9-nt heptaloops are ignored. All hairpin loops fall within a branch point value of 1.4 Å.

FIGURE 4.

Three-dimensional structure of loop 2737–2742 and residues 1561–1562 in Haloarcula marismortui-23S rRNA. RNA labeling is based on the scheme proposed by Leontis and Westhof (2001). Additional bases from the 5′ half of the 23S rRNA that form Watson–Crick pairs with bases in the loop are also shown.