Mutate-and-map profiling of the human CR4/5 domain indicates the presence of structural heterogeneity within the RNA junction region. (A) Systematic mutations were introduced at each base within the hTR CR4/5 domain as indicated (A → U, U → A, G → C, and C → U). The structure of each mutant was interrogated by SHAPE (1M7), and the resultant reactivity profiles were stacked to create a reactivity tapestry that permits visual comparison of the chemical reactivity at each nucleotide across all mutants. The red dashed line corresponds to the position of expected signal of enhanced reactivity at the site of the base substitution. Specific sites of enhanced reactivity (“release events”) are circled in red. Positions of validated base pairing interactions are highlighted in red in the secondary structure model shown to the right. Mutation positions with the P6 stem (blue arrows) and P6.1 stem (purple arrows) that induce large-scale changes in the reactivity patterns are indicated. (B) Summary of specific mutations and sites of correlated enhancements of chemical reactivity together with the positions of the CR4/5 base pairs that these data support. (C) Bootstrap support values are plotted in a base pair probability matrix represented in gray scale. High confidence stems give rise to dark and symmetric signals. Each of the RNA structure elements are annotated including noncanonical cross-junction clamps and an alternate P6.1 stem. (D) Representative alternative hTR CR4/5 junction structure predictions from mutate-and-map experiments.
