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Helix packing is critical for RNA tertiary structure formation, although the rules for helix–helix association within structured RNAs are largely unknown. Docking of the substrate helix into
the active site of the Tetrahymena group I ribozyme provides a model system to study this question. Using a novel chemogenetic method to analyze RNA structure in atomic detail, we report
that complementary sets of noncanonical base pairs (a G·U wobble pair and two consecutively stacked sheared A·A pairs) create an RNA helix packing motif that is essential for 5′-splice site
selection in the group I intron. This is likely to be a general motif for helix–helix interaction within the tertiary structures of many large RNAs.
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