A significant fraction of the bases in a folded,
structured RNA molecule participate in noncanonical base
pairing interactions, often in the context of internal
loops or multi-helix junction loops. The appearance of
each new high-resolution RNA structure provides welcome
data to guide efforts to understand and predict RNA 3D
structure, especially when the RNA in question is a functionally
conserved molecule. The recent publication of the crystal
structure of the “Loop E” region of bacterial
5S ribosomal RNA is such an event [Correll CC, Freeborn
B, Moore PB, Steitz TA, 1997, Cell 91:705–712].
In addition to providing more examples of already established
noncanonical base pairs, such as purine–purine sheared
pairings, trans-Hoogsteen UA, and GU wobble pairs,
the structure provides the first high-resolution views
of two new purine–purine pairings and a new GU pairing.
The goal of the present analysis is to expand the capabilities
of both chemical probing and phylogenetic analysis to predict
with greater accuracy the structures of RNA molecules.
First, in light of existing chemical probing data, we investigate
what lessons could be learned regarding the interpretation
of this widely used method of RNA structure probing. Then
we analyze the 3D structure with reference to molecular
phylogeny data (assuming conservation of function) to discover
what alternative base pairings are geometrically compatible
with the structure. The comparisons between previous modeling
efforts and crystal structures show that the intricate
involvements of ions and water molecules in the maintenance
of non-Watson–Crick pairs render the process of correctly
identifying the interacting sites in such pairs treacherous,
except in cases of trans-Hoogsteen A/U or sheared
A/G pairs for the adenine N1 site. The phylogenetic analysis
identifies A/A, A/C, A/U and C/A, C/C, and C/U pairings
isosteric with sheared A/G, as well as A/A and A/C pairings
isosteric with both G/U and G/G bifurcated pairings. Thus,
each non-Watson–Crick pair could be characterized
by a phylogenetic signature of variations between isosteric-like
pairings. In addition to the conservative changes, which
form a dictionary of pairings isosterically compatible
with those observed in the crystal structure, concerted
changes involving several base pairs also occur. The latter
covariations may indicate transitions between related but
distinctive motifs within the loop E of 5S ribosomal RNA.