Interaction of DsrA–rpoS in E. coli as a direct path energy landscape following RRIkinDP (Waldl et al. 2023) and comparison of different formation scenarios. RRIkinDP, a novel 2D structure-based tool for studying RNA–RNA interaction formation on direct paths, was used to generate the energy landscape of structures along folding pathways from a first interaction base pair to the full DsrA–rpoS interaction. To study interaction formation, RRIkinDP considers intermediate interactions formed by consecutive interaction base pairs. Each intermediate interaction is therefore defined by the first and last interaction pair. For visualization, intermediate states can therefore be arranged in an upper triangular matrix, indexed by two enclosing pairs on the x- and y-axis. Each cell of the matrix represents an intermediate interaction, with minimal interactions consisting of a single base pair on the diagonal and the full, maximal length, interaction in the upper right corner. Cell colors depict the free energy ΔG on a red-to-blue scale. For details, see Waldl et al. (2023). The predicted 2D interaction for DsrA–rpoS appears in the lower left triangle, presented in two ways: the interaction site above, with interior loops marked as (1) and (2), and a 2D representation of the complete interaction complex below. Using the energy landscape, we identified three energetically favorable start sites (highlighted in orange): within the loop of the first stem–loop (SL1), within the LR, and within the bulge of the second stem–loop (SL2), as shown in Figure 1. From these sites, we generated direct folding paths in 2D, which were subsequently embedded in 3D using the RRI-3D pipeline. The resulting 3D pathways were projected back into 2D and are depicted as black lines within the heatmap. In the path, moving diagonally corresponds to extending the interaction on both sides, while moving up or to the right extends the interaction by 1 bp on the left or right, respectively. Different linestyles represent different initial folds of the DsrA SL2 structure (see legend and Fig. 1).
